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Sample records for mature mdx mice

  1. Lack of Dystrophin Affects Bronchial Epithelium in mdx Mice.

    PubMed

    Morici, Giuseppe; Rappa, Francesca; Cappello, Francesco; Pace, Elisabetta; Pace, Andrea; Mudò, Giuseppa; Crescimanno, Grazia; Belluardo, Natale; Bonsignore, Maria R

    2016-10-01

    Mild exercise training may positively affect the course of Duchenne Muscular Dystrophy (DMD). Training causes mild bronchial epithelial injury in both humans and mice, but no study assessed the effects of exercise in mdx mice, a well known model of DMD. The airway epithelium was examined in mdx (C57BL/10ScSn-Dmdmdx) mice, and in wild type (WT, C57BL/10ScSc) mice either under sedentary conditions (mdx-SD, WT-SD) or during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days of training (5 d/wk for 6 weeks), epithelial morphology and markers of regeneration, apoptosis, and cellular stress were assessed. The number of goblet cells in bronchial epithelium was much lower in mdx than in WT mice under all conditions. At 30 days, epithelial regeneration (PCNA positive cells) was higher in EX than SD animals in both groups; however, at 45 days, epithelial regeneration decreased in mdx mice irrespective of training, and the percentage of apoptotic (TUNEL positive) cells was higher in mdx-EX than in WT-EX mice. Epithelial expression of HSP60 (marker of stress) progressively decreased, and inversely correlated with epithelial apoptosis (r = -0.66, P = 0.01) only in mdx mice. Lack of dystrophin in mdx mice appears associated with defective epithelial differentiation, and transient epithelial regeneration during mild exercise training. Hence, lack of dystrophin might impair repair in bronchial epithelium, with potential clinical consequences in DMD patients. J. Cell. Physiol. 231: 2218-2223, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868633

  2. Disease course in mdx:utrophin+/− mice: comparison of three mouse models of Duchenne muscular dystrophy

    PubMed Central

    McDonald, Abby A; Hebert, Sadie L; Kunz, Matthew D; Ralles, Steven J; McLoon, Linda K

    2015-01-01

    The mdx mouse model of Duchenne muscular dystrophy (DMD) is used to study disease mechanisms and potential treatments, but its pathology is less severe than DMD patients. Other mouse models were developed to more closely mimic the human disease based on knowledge that upregulation of utrophin has a protective effect in mdx muscle. An mdx:utrophin−/− (dko) mouse was created, which had a severe disease phenotype and a shortened life span. An mdx:utrophin+/− mouse was also created, which had an intermediate disease phenotype compared to the mdx and dko mice. To determine the usefulness of mdx:utrophin+/− mice for long-term DMD studies, limb muscle pathology and function were assessed across the life span of wild-type, mdx, mdx:utrophin+/−, and dko mice. Muscle function assessment, specifically grip duration and rotarod performance, demonstrated that mdx:utrophin+/− mice were weaker for a longer time than mdx mice. Mean myofiber area was smaller in mdx:utrophin+/− mice compared to mdx mice at 12 months. Mdx:utrophin+/− mice had a higher percentage of centrally nucleated myofibers compared to mdx mice at 6 and 12 months. Collagen I and IV density was significantly higher in mdx:utrophin+/− muscle compared to mdx at most ages examined. Generally, mdx:utrophin+/− mice showed an intermediate disease phenotype over a longer time course compared to the mdx and dko mice. While they do not genetically mirror human DMD, mdx:utrophin+/− mice may be a more useful animal model than mdx or dko mice for investigating long-term efficacy of potential treatments when fibrosis or muscle function is the focus. PMID:25921779

  3. A canine minidystrophin is functional and therapeutic in mdx mice.

    PubMed

    Wang, B; Li, J; Qiao, C; Chen, C; Hu, P; Zhu, X; Zhou, L; Bogan, J; Kornegay, J; Xiao, X

    2008-08-01

    Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder lacking a curative treatment. We wish to use the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog, a canine model of DMD, to investigate adeno-associated virus (AAV) vector-mediated minidystrophin gene therapy. The dog model is useful in evaluating vector dose requirement and immunological consequences owing to its large size and outbred nature. In this study, we have cloned and constructed a canine minidystrophin gene vector. Owing to limited availability of the GRMD dogs, here we first examined the functions and therapeutic effects of the canine minidystrophin in the mdx mouse model. We observed efficient minigene expression without cellular immune responses in mdx mice after AAV1-cMinidys vector intramuscular injection. We also observed restoration of the missing dystrophin-associated protein complex (DPC) onto the sarcolemma, including sarcoglycans and dystrobrevin, and a partial restoration of alpha-syntrophin and neural nitric oxide synthase (nNOS). In addition, minidystrophin treatment ameliorated dystrophic pathology, such as fibrosis and myofiber central nucleation (CN). CN remained minimal (<2%) after AAV injection in the neonatal mdx mice and was reduced from more than 75% to about 25% after AAV injection in adult mdx mice. Finally, in vivo cell membrane leakage test with Evans blue dye showed that the canine minidystrophin could effectively protect the myofiber plasma membrane integrity. Our results, thus, demonstrated the functionality and therapeutic potential of the canine minidystrophin and paved its way for further testing in the GRMD dog model. PMID:18432277

  4. Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice

    PubMed Central

    Markham, Bruce E.; Kernodle, Stace; Nemzek, Jean; Wilkinson, John E.; Sigler, Robert

    2015-01-01

    Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone’s effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients. PMID:26248188

  5. Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.

    PubMed

    Chamberlain, Jeffrey S; Metzger, Joseph; Reyes, Morayma; Townsend, DeWayne; Faulkner, John A

    2007-07-01

    Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the approximately 20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild-type C57BL/10 mice housed under specific pathogen-free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild-type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopathology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD. PMID:17360850

  6. Evidence of hypoxic tolerance in weak upper airway muscle from young mdx mice.

    PubMed

    Burns, David P; O'Halloran, Ken D

    2016-06-01

    Duchenne muscular dystrophy (DMD) is a genetic disease characterised by deficiency in the protein dystrophin. The respiratory system is weakened and patients suffer from sleep disordered breathing and hypoventilation culminating in periods of hypoxaemia. We examined the effects of an acute (6h) hypoxic stress on sternohyoid muscle function (representative pharyngeal dilator). 8 week old male, wild-type (WT; C57BL/10ScSnJ; n=18) and mdx (C57BL/10ScSn-Dmd(mdx)/J; n=16) mice were exposed to sustained hypoxia (FIO2=0.10) or normoxia. Muscle functional properties were examined ex vivo. Additional WT (n=5) and mdx (n=5) sternohyoid muscle was exposed to an anoxic challenge. Sternohyoid dysfunction was observed in mdx mice with significant reductions in force and power. Following exposure to the acute in vivo hypoxic stress, WT sternohyoid muscle showed evidence of functional impairment (reduced force, work and power). Conversely, mdx sternohyoid showed an apparent tolerance to the acute hypoxic stress. This tolerance was not maintained for mdx following a severe hypoxic stress. A dysfunctional upper airway muscle phenotype is present at 8 weeks of age in the mdx mouse, which may have implications for the control of airway patency in DMD. Hypoxic tolerance in mdx respiratory muscle is suggestive of adaptation to chronic hypoxia, which could be present due to respiratory morbidity. We speculate a role for hypoxia in mdx respiratory muscle morbidity. PMID:26691169

  7. Delayed Cardiomyopathy in Dystrophin Deficient mdx Mice Relies on Intrinsic Glutathione Resource

    PubMed Central

    Khouzami, Lara; Bourin, Marie-Claude; Christov, Christo; Damy, Thibaud; Escoubet, Brigitte; Caramelle, Philippe; Perier, Magali; Wahbi, Karim; Meune, Christophe; Pavoine, Catherine; Pecker, Françoise

    2010-01-01

    Oxidative stress contributes to the pathogenesis of Duchenne muscular dystrophy (DMD). Although they have been a model for DMD, mdx mice exhibit slowly developing cardiomyopathy. We hypothesized that disease process was delayed owing to the development of an adaptive mechanism against oxidative stress, involving glutathione synthesis. At 15 to 20 weeks of age, mdx mice displayed a 33% increase in blood glutathione levels compared with age-matched C57BL/6 mice. In contrast, cardiac glutathione content was similar in mdx and C57BL/6 mice as a result of the balanced increased expression of glutamate cysteine ligase catalytic and regulatory subunits ensuring glutathione synthesis in the mdx mouse heart, as well as increased glutathione peroxidase-1 using glutathione. Oral administration from 10 weeks of age of the glutamate cysteine ligase inhibitor, l-buthionine(S,R)-sulfoximine (BSO, 5 mmol/L), led to a 33% and 50% drop in blood and cardiac glutathione, respectively, in 15- to 20-week-old mdx mice. Moreover, 20-week-old BSO-treated mdx mice displayed left ventricular hypertrophy associated with diastolic dysfunction, discontinuities in β-dystroglycan expression, micronecrosis and microangiopathic injuries. Examination of the glutathione status in four DMD patients showed that three displayed systemic glutathione deficiency as well. In conclusion, low glutathione resource hastens the onset of cardiomyopathy linked to a defect in dystrophin in mdx mice. This is relevant to the glutathione deficiency that DMD patients may suffer. PMID:20696779

  8. Erythropoietin reduces the expression of myostatin in mdx dystrophic mice.

    PubMed

    Feder, D; Rugollini, M; Santomauro, A; Oliveira, L P; Lioi, V P; Santos, R dos; Ferreira, L G; Nunes, M T; Carvalho, M H; Delgado, P O; Carvalho, A A S; Fonseca, F L A

    2014-11-01

    Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO = 0.60 ± 0.11, control = 1.07 ± 0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO = 0.95 ± 0.14, control = 1.05 ± 0.16) and TNF-α (rhEPO = 0.73 ± 0.20, control = 1.01 ± 0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle. PMID:25296358

  9. Reduction in mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised compared with sedentary mdx mice

    PubMed Central

    Fontana, Simona; Schillaci, Odessa; Frinchi, Monica; Giallombardo, Marco; Morici, Giuseppe; Liberto, Valentina Di; Alessandro, Riccardo; De Leo, Giacomo; Perciavalle, Vincenzo; Belluardo, Natale; Mudò, Giuseppa

    2015-01-01

    In our recent study was shown a significant recovery of damaged skeletal muscle of mice with X-linked muscular dystrophy (mdx) following low-intensity endurance exercise, probably by reducing the degeneration of dystrophic muscle. Consequently, in the present work, we aimed to identify proteins involved in the observed reduction in degenerating fibres. To this end, we used proteomic analysis to evaluate changes in the protein profile of quadriceps dystrophic muscles of exercised compared with sedentary mdx mice. Four protein spots were found to be significantly changed and were identified as three isoforms of carbonic anhydrase 3 (CA3) and superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were significantly up-regulated in quadriceps of sedentary mdx mice and were completely restored to wild–type (WT) mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Protein levels of SODC were down-regulated in quadriceps of sedentary mdx mice and were significantly restored to WT mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Western blot data were in agreement with those obtained using proteomic analysis and revealed the presence of one more CA3 isoform that was significantly changed. Based on data found in the present study, it seems that low-intensity endurance exercise may in part contribute to reduce cell degeneration process in mdx muscles, by counteracting oxidative stress. PMID:26182375

  10. Arginine butyrate per os protects mdx mice against cardiomyopathy, kyphosis and changes in axonal excitability.

    PubMed

    Vianello, Sara; Bouyon, Sophie; Benoit, Evelyne; Sebrié, Catherine; Boerio, Delphine; Herbin, Marc; Roulot, Morgane; Fromes, Yves; de la Porte, Sabine

    2014-11-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by lack of dystrophin, a sub-sarcolemmal protein, which leads to dramatic muscle deterioration. We studied in mdx mice, the effects of oral administration of arginine butyrate (AB), a compound currently used for the treatment of sickle cell anemia in children, on cardiomyopathy, vertebral column deformation and electromyographic abnormalities. Monthly follow-up by echocardiography from the 8th month to the 14th month showed that AB treatment protected the mdx mice against drastic reduction (20-23%) of ejection fraction and fractional shortening, and also against the ≈20% ventricular dilatation and 25% cardiac hypertrophy observed in saline-treated mdx mice. The phenotypic improvement was corroborated by the decrease in serum CK level and by better fatigue resistance. Moreover, AB treatment protected against the progressive spinal deformity observed in mdx mice, another similarity with DMD patients. The value of the kyphosis index in AB-treated mice reached 94% of the value in C57BL/10 mice. Finally, axonal excitability parameters such as the membrane resting potential, the threshold and amplitude of the action potential, the absolute and relative refractory periods and the supernormal and subnormal periods, recorded from caudal and plantar muscles in response to excitability tests, that were modified in saline-treated mdx mice were not significantly changed, compared with wild-type animals, in AB-treated mdx mice. All of these results suggest that AB could be a potential treatment for DMD patients. PMID:25167832

  11. Gastric emptying, small intestinal transit and fecal output in dystrophic (mdx) mice.

    PubMed

    Mulè, Flavia; Amato, Antonella; Serio, Rosa

    2010-01-01

    Duchenne muscular dystrophy (DMD), which results from deficiency in dystrophin, a sarcolemma protein of skeletal, cardiac and smooth muscle, is characterized by progressive striated muscle degeneration, but various gastrointestinal clinical manifestations have been observed. The aim was to evaluate the possible impact of the dystrophin loss on the gastrointestinal propulsion in mdx mice (animal model for DMD). The gastric emptying of a carboxymethyl cellulose/phenol red dye non-nutrient meal was not significantly different at 20 min from gavaging between wild-type and mdx mice. The intestinal transit and the fecal output were significantly decreased in mdx versus normal animals, although the length of the intestine was similar in both animals. The present results provide evidence for motor intestinal alterations in mdx mice in in vivo conditions. PMID:19784719

  12. Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice.

    PubMed

    Yajima, Hiroshi; Kawakami, Kiyoshi

    2016-08-01

    Muscle regeneration is an important process for skeletal muscle growth and recovery. Repair of muscle damage is exquisitely programmed by cellular mechanisms inherent in myogenic stem cells, also known as muscle satellite cells. We demonstrated previously the involvement of homeobox transcription factors, SIX1, SIX4 and SIX5, in the coordinated proliferation and differentiation of isolated satellite cells in vitro. However, their roles in adult muscle regeneration in vivo remain elusive. To investigate SIX4 and SIX5 functions during muscle regeneration, we introduced knockout alleles of Six4 and Six5 into an animal model of Duchenne Muscular Dystrophy (DMD), mdx (Dmd(mdx) /Y) mice, characterized by frequent degeneration-regeneration cycles in muscles. A lower number of small myofibers, higher number of thick ones and lower serum creatine kinase and lactate dehydrogenase activities were noted in 50-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice than Dmd(mdx) /Y mice, indicating improvement of dystrophic phenotypes of Dmd(mdx) /Y mice. Higher proportions of cells positive for MYOD1 and MYOG (markers of regenerating myonuclei) and SIX1 (a marker of regenerating myoblasts and newly regenerated myofibers) in 12-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice suggested enhanced regeneration, compared with Dmd(mdx) /Y mice. Although grip strength was comparable in Six4(+/-) 5(+/-) Dmd(mdx) /Y and Dmd(mdx) /Y mice, treadmill exercise did not induce muscle weakness in Six4(+/-) 5(+/-) Dmd(mdx) /Y mice, suggesting higher regeneration capacity. In addition, Six4(+/-) 5(+/-) Dmd(mdx) /Y mice showed 33.8% extension of life span. The results indicated that low Six4 and Six5 gene dosage improved dystrophic phenotypes of Dmd(mdx) /Y mice by enhancing muscle regeneration, and suggested that SIX4 and SIX5 are potentially useful de novo targets in therapeutic applications against muscle disorders, including DMD. PMID:27224259

  13. Activation of Wnt3a signaling promotes myogenic differentiation of mesenchymal stem cells in mdx mice

    PubMed Central

    Shang, Yan-chang; Wang, Shu-hui; Xiong, Fu; Peng, Fu-ning; Liu, Zhen-shan; Geng, Jia; Zhang, Cheng

    2016-01-01

    Aim: Duchenne muscular dystrophy (DMD) is an X-linked genetic muscular disorder with no effective treatment at present. Mesenchymal stem cell (MSC) transplantation has been used to treat DMD, but the efficiency is low. Our previous studies show that activation of Wnt3a signaling promotes myogenic differentiation of MSCs in vitro. Here we report an effective MSC transplantation therapy in mdx mice by activation of Wnt3a signaling. Methods: MSCs were isolated from mouse bone marrow, and pretreated with Wnt3a-conditioned medium (Wnt3a-CM), then transplanted into mdx mice. The recipient mice were euthanized at 4, 8, 12, 16 weeks after the transplantation, and muscle pathological changes were examined. The expression of dystrophin in muscle was detected using immunofluorescence staining, RT-PCR and Western blotting. Results: Sixteen weeks later, transplantation of Wnt3a-pretreated MSCs in mdx mice improved the characteristics of dystrophic muscles evidenced by significant reductions in centrally nucleated myofibers, the variability range of cross-sectional area (CSA) and the connective tissue area of myofibers. Furthermore, transplantation of Wnt3a-pretreated MSCs in mdx mice gradually and markedly increased the expression of dystrophin in muscle, and improved the efficiency of myogenic differentiation. Conclusion: Transplantation of Wnt3a-pretreated MSCs in mdx mice results in long-term amelioration of the dystrophic phenotype and restores dystrophin expression in muscle. The results suggest that Wnt3a may be a promising candidate for the treatment of DMD. PMID:27133298

  14. Muscle genome-wide expression profiling during disease evolution in mdx mice.

    PubMed

    Marotta, Mario; Ruiz-Roig, Claudia; Sarria, Yaris; Peiro, Jose Luis; Nuñez, Fatima; Ceron, Julian; Munell, Francina; Roig-Quilis, Manuel

    2009-04-10

    Mdx mice show a milder phenotype than Duchenne patients despite bearing an analogous genetic defect. Our aim was to sort out genes, differentially expressed during the evolution of skeletal muscle mdx mouse disease, to elucidate the mechanisms by which these animals overcome the lack of dystrophin. Genome-wide microarray-based gene expression analysis was carried out at 3 wk and 1.5 and 3 mo of life. Candidate genes were selected by comparing: 1) mdx vs. controls at each point in time, and 2) mdx mice and 3) control mice among the three points in time. The first analysis showed a strong upregulation (96%) of inflammation-related genes and in >75% of genes related to cell adhesion, muscle structure/regeneration, and extracellular matrix remodeling during mdx disease evolution. Lgals3, Postn, Ctss, and Sln genes showed the strongest variations. The analysis performed among points in time demonstrated significant changes in Ecm1, Spon1, Thbs1, Csrp3, Myo10, Pde4b, and Adamts-5 exclusively during mdx mice lifespan. RT-PCR analysis of Postn, Sln, Ctss, Thbs1, Ecm1, and Adamts-5 expression from 3 wk to 9 mo, confirmed microarray data and demonstrated variations beyond 3 mo of age. A high-confidence functional network analysis demonstrated a strong relationship between them and showed two main subnetworks, having Dmd-Utrn-Myo10 and Adamts5-Thbs1-Spon1-Postn as principal nodes, which are functionally linked to Abca1, Actn4, Crebbp, Csrp3, Lama1, Lama3, Mical2, Mical3, Myf6, Pxn, and Sparc genes. Candidate genes may participate in the decline of muscle necrosis in mdx mice and could be considered potential therapeutic targets for Duchenne patients. PMID:19223608

  15. Aquapuncture Using Stem Cell Therapy to Treat Mdx Mice

    PubMed Central

    Esper, Greyson Vitor Zanatta; Pignatari, Graciela Conceição; Rodrigues, Marcio Nogueira; Bertagnon, Heloisa Godoi; Fernandes, Isabella Rodrigues; Nascimento, Nanci; Tabosa, Angela Maria Florencio; Beltrão-Braga, Patrícia Cristina Baleeiro; Miglino, Maria Angelica

    2015-01-01

    Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to absence or decrease of dystrophin protein generating progressive muscle degeneration. Cell therapy using mesenchymal stem cell (MSC) has been described as a treatment to DMD. In this work, MSC derived from deciduous teeth, called stem cells from human exfoliated deciduous teeth (SHED), were injected in acupoint as an alternative therapy to minimize muscle degeneration in twenty-two mdx mice. The treatment occurred three times with intervals of 21 days, and animals were analyzed four times: seven days prior treatment (T-7); 10 days after first treatment (T10); 10 days after second treatment (T31); and 10 days after third treatment (T52). Animals were evaluated by wire test for estimate strength and blood was collected to perform a creatinine phosphokinase analysis. After euthanasia, cranial tibial muscles were collected and submitted to histological and immunohistochemistry analyses. Treated groups presented improvement of strength and reduced creatinine phosphokinase levels. Also, a slight dystrophin increase was observed in tibial cranial muscle when aquapuncture was associated SHED. All therapies have minimized muscle degeneration, but the association of aquapuncture with SHED appears to have better effect, reducing muscle damage, suggesting a therapeutic value. PMID:26074983

  16. Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

    PubMed Central

    Williams, D A; Head, S I; Lynch, G S; Stephenson, D G

    1993-01-01

    1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which

  17. Losartan decreases cardiac muscle fibrosis and improves cardiac function in dystrophin-deficient mdx mice.

    PubMed

    Spurney, Christopher F; Sali, Arpana; Guerron, Alfredo D; Iantorno, Micaela; Yu, Qing; Gordish-Dressman, Heather; Rayavarapu, Sree; van der Meulen, Jack; Hoffman, Eric P; Nagaraju, Kanneboyina

    2011-03-01

    Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmd(mdx)/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice. PMID:21304057

  18. Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene.

    PubMed

    Matsumura, K; Lee, C C; Caskey, C T; Campbell, K P

    1993-04-12

    Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extracellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex. PMID:8462697

  19. Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice

    PubMed Central

    Williams, McRae W.; Bloch, Robert J.

    1999-01-01

    We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. β-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, β-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, β-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain β-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with β-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including α-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle. PMID:10087268

  20. Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice.

    PubMed

    Latroche, Claire; Matot, Béatrice; Martins-Bach, Aurea; Briand, David; Chazaud, Bénédicte; Wary, Claire; Carlier, Pierre G; Chrétien, Fabrice; Jouvion, Grégory

    2015-09-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease, caused by an absence of dystrophin, inevitably leading to death. Although muscle lesions are well characterized, blood vessel alterations that may have a major impact on muscle regeneration remain poorly understood. Our aim was to elucidate alterations of the vascular network organization, taking advantage of Flk1(GFP/+) crossed with mdx mice (model for human DMD where all blood vessels express green fluorescent protein) and functional repercussions using in vivo nuclear magnetic resonance, combining arterial spin-labeling imaging of perfusion, and (31)P-spectroscopy of phosphocreatine kinetics. For the first time, our study focused on old (12-month-old) mdx mice, displaying marked chronic muscle lesions, similar to the lesions observed in human DMD, in comparison to young-adult (3-month-old) mdx mice displaying only mild muscle lesions with no fibrosis. By using an original approach combining a specific animal model, state-of-the-art histology/morphometry techniques, and functional nuclear magnetic resonance, we demonstrated that the microvascular system is almost normal in young-adult in contrast to old mdx mice, displaying marked microvessel alterations, and the functional repercussions on muscle perfusion and bioenergetics after a hypoxic stress vary depending on stage of pathology. This original approach clarifies disease evolution and paves the way for setting up new diagnostic markers or therapeutic strategies. PMID:26193666

  1. Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice

    PubMed Central

    Huynh, Tony; Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Heier, Christopher R; Van Der Meulen, Jack H; Yu, Qing; Harris, Mark; Nolan, Christopher J; Haegeman, Guy; Grounds, Miranda D; Nagaraju, Kanneboyina

    2014-01-01

    The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2Kb-tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective

  2. Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice

    PubMed Central

    Pelosi, Laura; Berardinelli, Maria Grazia; Forcina, Laura; Spelta, Elisa; Rizzuto, Emanuele; Nicoletti, Carmine; Camilli, Carlotta; Testa, Erika; Catizone, Angela; De Benedetti, Fabrizio; Musarò, Antonio

    2015-01-01

    Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD. PMID:26251044

  3. Short Telomeres and Stem Cell Exhaustion Model Duchenne Muscular Dystrophy in mdx/mTR Mice

    PubMed Central

    Sacco, Alessandra; Mourkioti, Foteini; Tran, Rose; Choi, Jinkuk; Llewellyn, Michael; Kraft, Peggy; Shkreli, Marina; Delp, Scott; Pomerantz, Jason H.; Artandi, Steven E.; Blau, Helen M.

    2010-01-01

    Summary In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC) and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity, and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results in part from a cell-autonomous failure of MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD. PMID:21145579

  4. Fetal muscle-derived cells can repair dystrophic muscles in mdx mice

    SciTech Connect

    Auda-Boucher, Gwenola; Rouaud, Thierry; Lafoux, Aude; Levitsky, Dmitri; Huchet-Cadiou, Corinne; Feron, Marie; Guevel, Laetitia; Talon, Sophie; Fontaine-Perus, Josiane; Gardahaut, Marie-France . E-mail: Marie-France.Gardahaut@univ-nantes.fr

    2007-03-10

    We have previously reported that CD34{sup +} cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP{sup +}/CD34{sup +} cells or desmin{sup +}/{sup -}LacZ/CD34{sup +} cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

  5. Effect of injecting primary myoblasts versus putative muscle-derived stem cells on mass and force generation in mdx mice.

    PubMed

    Mueller, Gunhild M; O'Day, Terry; Watchko, Jon F; Ontell, Marcia

    2002-06-10

    It is well established that the injection of normal myoblasts or of muscle-derived stem cells (MDSCs) into the muscle of dystrophin-deficient mdx mice results in the incorporation of a number of donor myoblasts into the host muscle. However, the effect of the injected exogenous cells on mdx muscle mass and functional capacity has not been evaluated. This study evaluates the mass and functional capacity of the extensor digitorum longus (EDL) muscles of adult, male mdx mice that received intramuscular injections of primary myoblasts or of MDSCs (isolated by a preplating technique; Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J., J. Cell Biol. 1998;142:1257-1267) derived from normal mice. Evaluations were made 9 weeks after cell transplantation. Uninjected mdx EDL muscles have a mass 50% greater than that of age-matched C57BL/10J (normal) EDL muscles. Injections of either primary myoblasts or MDSCs have no effect on the mass of mdx EDL muscles. EDL muscles of mdx mice generate 43% more absolute twitch tension and 43% less specific tetanic tension then do EDL muscles of C57BL/10J mice. However, the absolute tetanic and specific twitch tension of mdx and C57BL/10J EDL muscles are similar. Injection of either primary myoblasts or MDSCs has no effect on the absolute or specific twitch and tetanic tensions of mdx muscle. Approximately 25% of the myofibers in mdx EDL muscles that received primary myoblasts react positively with antibody to dystrophin. There is no significant difference in the number of dystrophin-positive myofibers when MDSCs are injected. Regardless of the source of donor cells, dystrophin is limited to short distances (60-900 microm) along the length of the myofibers. This may, in part, explain the failure of cellular therapy to alter the contractile properties of murine dystrophic muscle. PMID:12067441

  6. Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

    PubMed

    Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie M R; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi

    2012-08-21

    Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model. PMID:22869723

  7. Rapid depletion of muscle progenitor cells in dystrophic mdx/utrophin−/− mice

    PubMed Central

    Lu, Aiping; Poddar, Minakshi; Tang, Ying; Proto, Jonathan D.; Sohn, Jihee; Mu, Xiaodong; Oyster, Nicholas; Wang, Bing; Huard, Johnny

    2014-01-01

    Duchenne muscular dystrophy (DMD) patients lack dystrophin from birth; however, muscle weakness becomes apparent only at 3–5 years of age, which happens to coincide with the depletion of the muscle progenitor cell (MPC) pools. Indeed, MPCs isolated from older DMD patients demonstrate impairments in myogenic potential. To determine whether the progression of muscular dystrophy is a consequence of the decline in functional MPCs, we investigated two animal models of DMD: (i) dystrophin-deficient mdx mice, the most commonly utilized model of DMD, which has a relatively mild dystrophic phenotype and (ii) dystrophin/utrophin double knock-out (dKO) mice, which display a similar histopathologic phenotype to DMD patients. In contrast to age-matched mdx mice, we observed that both the number and regeneration potential of dKO MPCs rapidly declines during disease progression. This occurred in MPCs at both early and late stages of myogenic commitment. In fact, early MPCs isolated from 6-week-old dKO mice have reductions in proliferation, resistance to oxidative stress and multilineage differentiation capacities compared with age-matched mdx MPCs. This effect may potentially be mediated by fibroblast growth factor overexpression and/or a reduction in telomerase activity. Our results demonstrate that the rapid disease progression in the dKO model is associated, at least in part, with MPC depletion. Therefore, alleviating MPC depletion could represent an approach to delay the onset of the histopathologies associated with DMD patients. PMID:24781208

  8. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice

    PubMed Central

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J. A.; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose–fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  9. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice.

    PubMed

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J A; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose-fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  10. Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice.

    PubMed

    Quinn, James L; Huynh, Tony; Uaesoontrachoon, Kitipong; Tatem, Kathleen; Phadke, Aditi; Van der Meulen, Jack H; Yu, Qing; Nagaraju, Kannaboyina

    2013-01-01

    Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. PMID:24270550

  11. Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice

    PubMed Central

    Quinn, James L; Huynh, Tony; Uaesoontrachoon, Kitipong; Tatem, Kathleen; Phadke, Aditi; Van der Meulen, Jack H; Yu, Qing; Nagaraju, Kannaboyina

    2013-01-01

    Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. PMID:24270550

  12. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

    PubMed

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  13. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  14. Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice.

    PubMed

    Huynh, Tony; Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Heier, Christopher R; Van Der Meulen, Jack H; Yu, Qing; Harris, Mark; Nolan, Christopher J; Haegeman, Guy; Grounds, Miranda D; Nagaraju, Kanneboyina

    2013-10-01

    The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective

  15. Nifedipine treatment reduces resting calcium concentration, oxidative and apoptotic gene expression, and improves muscle function in dystrophic mdx mice.

    PubMed

    Altamirano, Francisco; Valladares, Denisse; Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R; Allen, Paul D; Jaimovich, Enrique

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca(2+)]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+)]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+)]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox)/p47(phox) NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+)]r in mdx skeletal muscle cells. The results in this work open new perspectives

  16. Nifedipine Treatment Reduces Resting Calcium Concentration, Oxidative and Apoptotic Gene Expression, and Improves Muscle Function in Dystrophic mdx Mice

    PubMed Central

    Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R.; Allen, Paul D.; Jaimovich, Enrique

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca2+]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca2+]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca2+]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91phox/p47phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca2+]r in mdx skeletal muscle cells. The results in this work open new perspectives towards

  17. Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice

    PubMed Central

    Mozzetta, Chiara; Consalvi, Silvia; Saccone, Valentina; Tierney, Matthew; Diamantini, Adamo; Mitchell, Kathryn J; Marazzi, Giovanna; Borsellino, Giovanna; Battistini, Luca; Sassoon, David; Sacco, Alessandra; Puri, Pier Lorenzo

    2013-01-01

    HDAC inhibitors (HDACi) exert beneficial effects in mdx mice, by promoting endogenous regeneration; however, the cellular determinants of HDACi activity on dystrophic muscles have not been determined. We show that fibroadipogenic progenitors (FAP) influence the regeneration potential of satellite cells during disease progression in mdx mice and mediate HDACi ability to selectively promote regeneration at early stages of disease. FAPs from young mdx mice promote, while FAPs from old mdx mice repress, satellite cell-mediated formation of myotubes. In young mdx mice HDACi inhibited FAP adipogenic potential, while enhancing their ability to promote differentiation of adjacent satellite cells, through upregulation of the soluble factor follistatin. By contrast, FAPs from old mdx mice were resistant to HDACi-mediated inhibition of adipogenesis and constitutively repressed satellite cell-mediated formation of myotubes. We show that transplantation of FAPs from regenerating young muscles restored HDACi ability to increase myofibre size in old mdx mice. These results reveal that FAPs are key cellular determinants of disease progression in mdx mice and mediate a previously unappreciated stage-specific beneficial effect of HDACi in dystrophic muscles. PMID:23505062

  18. Myostatin/activin blocking combined with exercise reconditions skeletal muscle expression profile of mdx mice.

    PubMed

    Kainulainen, Heikki; Papaioannou, Konstantinos G; Silvennoinen, Mika; Autio, Reija; Saarela, Janne; Oliveira, Bernardo M; Nyqvist, Miro; Pasternack, Arja; 't Hoen, Peter A C; Kujala, Urho M; Ritvos, Olli; Hulmi, Juha J

    2015-01-01

    Duchenne muscular dystrophy is characterized by muscle wasting and decreased aerobic metabolism. Exercise and blocking of myostatin/activin signaling may independently or combined counteract muscle wasting and dystrophies. The effects of myostatin/activin blocking using soluble activin receptor-Fc (sActRIIB-Fc) administration and wheel running were tested alone or in combination for 7 weeks in dystrophic mdx mice. Expression microarray analysis revealed decreased aerobic metabolism in the gastrocnemius muscle of mdx mice compared to healthy mice. This was not due to reduced home-cage physical activity, and was further downregulated upon sActRIIB-Fc treatment in enlarged muscles. However, exercise activated pathways of aerobic metabolism and counteracted the negative effects of sActRIIB-Fc. Exercise and sActRIIB-Fc synergistically increased expression of major urinary protein, but exercise blocked sActRIIB-Fc induced phosphorylation of STAT5 in gastrocnemius muscle. In conclusion, exercise alone or in combination with myostatin/activin blocking corrects aerobic gene expression profiles of dystrophic muscle toward healthy wild type mice profiles. PMID:25304272

  19. The Effects of Experimental Sleep Apnea on Cardiac and Respiratory Functions in 6 and 18 Month Old Dystrophic (mdx) Mice.

    PubMed

    Chaudhari, Milind R; Fallavollita, James A; Farkas, Gaspar A

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal disease where over 90% of patients succumb to respiratory or cardiac failure. Sleep apnea and sleep disordered breathing (SDB) are noted in a plurality of DMD patients, and the resulting nocturnal episodic hypoxia (EH) cannot be ruled out as a contributing factor to cardiac and respiratory dysfunction. In this study, we investigated the impact of long-term episodic hypoxia, which mimics the cyclic hypoxia seen in sleep apnea, on cardiac and respiratory function in a murine model of DMD (mdx mice). Since the severity and prevalence of sleep apnea in DMD increases with age, we studied the impact of EH on young (6-month) and on older (18-month) mdx mice. Mice were either exposed for 12 weeks to EH (8 hours/day, 5 days/week) or to room air. We noted a significant increase in left ventricular (LV) dilatation (transthoracic echocardiography) on EH exposure in both age groups, but reduced LV contractility was seen only in 6-month old mice. With EH exposure, an increased fibrosis (hydroxyproline) was noted in both cardiac and diaphragm muscle in 18-month but not 6-month old mice. No significant change in relative diaphragm strength (in-vitro) was noted on EH exposure in 18-month old mice. In contrast, EH exposed 6-month old mice showed a significant increase in relative diaphragm strength. EH exposure did not result in any significant change in ventilatory parameters (barometric plethysmography) in awake 6-month old mdx mice. In contrast, 18-month old mdx mice showed considerable ventilatory dysfunction, consistent with reduced ventilatory reserve. Our findings highlight that sleep apnea impacts respiratory and cardiac function in muscular dystrophy, and that EH can have divergent effects on both systems. To our knowledge, this is the first comprehensive study to investigate the impact of EH on cardiac and respiratory function in mdx mice. PMID:26808526

  20. The Effects of Experimental Sleep Apnea on Cardiac and Respiratory Functions in 6 and 18 Month Old Dystrophic (mdx) Mice

    PubMed Central

    Fallavollita, James A.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal disease where over 90% of patients succumb to respiratory or cardiac failure. Sleep apnea and sleep disordered breathing (SDB) are noted in a plurality of DMD patients, and the resulting nocturnal episodic hypoxia (EH) cannot be ruled out as a contributing factor to cardiac and respiratory dysfunction. In this study, we investigated the impact of long-term episodic hypoxia, which mimics the cyclic hypoxia seen in sleep apnea, on cardiac and respiratory function in a murine model of DMD (mdx mice). Since the severity and prevalence of sleep apnea in DMD increases with age, we studied the impact of EH on young (6-month) and on older (18-month) mdx mice. Mice were either exposed for 12 weeks to EH (8 hours/day, 5 days/week) or to room air. We noted a significant increase in left ventricular (LV) dilatation (transthoracic echocardiography) on EH exposure in both age groups, but reduced LV contractility was seen only in 6-month old mice. With EH exposure, an increased fibrosis (hydroxyproline) was noted in both cardiac and diaphragm muscle in 18-month but not 6-month old mice. No significant change in relative diaphragm strength (in-vitro) was noted on EH exposure in 18-month old mice. In contrast, EH exposed 6-month old mice showed a significant increase in relative diaphragm strength. EH exposure did not result in any significant change in ventilatory parameters (barometric plethysmography) in awake 6-month old mdx mice. In contrast, 18-month old mdx mice showed considerable ventilatory dysfunction, consistent with reduced ventilatory reserve. Our findings highlight that sleep apnea impacts respiratory and cardiac function in muscular dystrophy, and that EH can have divergent effects on both systems. To our knowledge, this is the first comprehensive study to investigate the impact of EH on cardiac and respiratory function in mdx mice. PMID:26808526

  1. Prevention of exercised induced cardiomyopathy following Pip-PMO treatment in dystrophic mdx mice

    PubMed Central

    Betts, Corinne A.; Saleh, Amer F.; Carr, Carolyn A.; Hammond, Suzan M.; Coenen-Stass, Anna M. L.; Godfrey, Caroline; McClorey, Graham; Varela, Miguel A.; Roberts, Thomas C.; Clarke, Kieran; Gait, Michael J.; Wood, Matthew J. A.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder caused by mutations in the Dmd gene. In addition to skeletal muscle wasting, DMD patients develop cardiomyopathy, which significantly contributes to mortality. Antisense oligonucleotides (AOs) are a promising DMD therapy, restoring functional dystrophin protein by exon skipping. However, a major limitation with current AOs is the absence of dystrophin correction in heart. Pip peptide-AOs demonstrate high activity in cardiac muscle. To determine their therapeutic value, dystrophic mdx mice were subject to forced exercise to model the DMD cardiac phenotype. Repeated peptide-AO treatments resulted in high levels of cardiac dystrophin protein, which prevented the exercised induced progression of cardiomyopathy, normalising heart size as well as stabilising other cardiac parameters. Treated mice also exhibited significantly reduced cardiac fibrosis and improved sarcolemmal integrity. This work demonstrates that high levels of cardiac dystrophin restored by Pip peptide-AOs prevents further deterioration of cardiomyopathy and pathology following exercise in dystrophic DMD mice. PMID:25758104

  2. Prevention of exercised induced cardiomyopathy following Pip-PMO treatment in dystrophic mdx mice.

    PubMed

    Betts, Corinne A; Saleh, Amer F; Carr, Carolyn A; Hammond, Suzan M; Coenen-Stass, Anna M L; Godfrey, Caroline; McClorey, Graham; Varela, Miguel A; Roberts, Thomas C; Clarke, Kieran; Gait, Michael J; Wood, Matthew J A

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder caused by mutations in the Dmd gene. In addition to skeletal muscle wasting, DMD patients develop cardiomyopathy, which significantly contributes to mortality. Antisense oligonucleotides (AOs) are a promising DMD therapy, restoring functional dystrophin protein by exon skipping. However, a major limitation with current AOs is the absence of dystrophin correction in heart. Pip peptide-AOs demonstrate high activity in cardiac muscle. To determine their therapeutic value, dystrophic mdx mice were subject to forced exercise to model the DMD cardiac phenotype. Repeated peptide-AO treatments resulted in high levels of cardiac dystrophin protein, which prevented the exercised induced progression of cardiomyopathy, normalising heart size as well as stabilising other cardiac parameters. Treated mice also exhibited significantly reduced cardiac fibrosis and improved sarcolemmal integrity. This work demonstrates that high levels of cardiac dystrophin restored by Pip peptide-AOs prevents further deterioration of cardiomyopathy and pathology following exercise in dystrophic DMD mice. PMID:25758104

  3. Ventilation during air breathing and in response to hypercapnia in 5 and 16 month-old mdx and C57 mice

    PubMed Central

    Gayraud, Jérome; Matécki, Stefan; Hnia, Karim; Mornet, Dominique; Préfaut, Christian; Mercier, Jacques; Michel, Alain; Ramonatxo, Michèle

    2007-01-01

    Previous studies have shown a blunted ventilatory response to hypercapnia in mdx mice older than 7 months. We test the hypothesis that in the mdx mice ventilatory response changes with age, concomitantly with the increased functional impairment of the respiratory muscles. We thus studied the ventilatory response to CO2 in 5 and 16 month-old mdx and C57BL10 mice (n = 8 for each group). Respiratory rate (RR), tidal volume (VT), and minute ventilation (VE) were measured, using whole-body plethysmography, during air breathing and in response to hypercapnia (3, 5 and 8% CO2). The ventilatory protocol was completed by histological analysis of the diaphragm and intercostals muscles. During air breathing, the 16 month-old mdx mice showed higher RR and, during hypercapnia (at 8% CO2 breathing), significantly lower RR (226 ± 26 vs. 270 ± 21 breaths/min) and VE (1.81 ± 0.35 vs. 3.96 ± 0.59 ml min−1 g−1)(P < 0.001) in comparison to C57BL10 controls. On the other hand, 5 month-old C57BL10 and mdx mice did not present any difference in their ventilatory response to air breathing and to hypercapnia. In conclusion, this study shows similar ventilation during air breathing and in response to hypercapnia in the 5 month-old mdx and control mice, in spite of significant pathological structural changes in the respiratory muscles of the mdx mice. However in the 16 month-old mdx mice we observed altered ventilation under air and blunted ventilation response to hypercapnia compared to age-matched control mice. Ventilatory response to hypercapnia thus changes with age in mdx mice, in line with the increased histological damage of their respiratory muscles. PMID:17431804

  4. CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.

    PubMed

    Xu, Li; Park, Ki Ho; Zhao, Lixia; Xu, Jing; El Refaey, Mona; Gao, Yandi; Zhu, Hua; Ma, Jianjie; Han, Renzhi

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD. PMID:26449883

  5. Polyethylenimine-modified pluronics (PCMs) improve morpholino oligomer delivery in cell culture and dystrophic mdx mice.

    PubMed

    Wang, Mingxing; Wu, Bo; Lu, Peijuan; Cloer, Caryn; Tucker, Jay D; Lu, Qilong

    2013-01-01

    We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2-6 k, with hydrophilic-lipophilic balance (HLB) 7-23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system. Application of optimized formulations of PCMs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. Consistent with our observations in vitro, optimization of molecular size and the HLB of pluronics are important factors for PCMs to achieve enhanced PMO delivery in vivo. Observed cytotoxicity of the PCMs was lower than Endo-porter and PEI 25 k. Tissue toxicity of PCMs in muscle was not clearly detected with the concentrations used, indicating the potential of the PCMs as effective and safe PMO carriers for treating diseases such as muscular dystrophy. PMID:23164938

  6. Effects of Mechanical Overloading on the Properties of Soleus Muscle Fibers, with or without Damage in MDX and Wild Type Mice

    NASA Astrophysics Data System (ADS)

    Terada, Masahiro; Kawano, Fuminori; Ohira, Takashi; Oke, Yoshihiko; Nakai, Naoya; Ohira, Yoshinobu

    2008-06-01

    Effects of mechanical overloading on the characteristics of regenerating or not-regenerating soleus muscle fibers were studied. The muscle fibers of mdx mice were characterized by the localization of myonuclei. Muscle damage was also induced in wild type (WT) mice by injection of cardiotoxin (CTX) into soleus muscle. Overloading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mice by removing the distal tendons of plantaris and gastrocnemius muscles. The contralateral muscle served as the normal control. These animals were then allowed ambulation recovery in the cage. Central myonuclei were noted in many fibers of mdx and CTX-injected mice with or without overloading. In general, the fibers with central nuclei were considered as regenerating fibers. The fibers with more central nuclei were increased in mdx mice, but the fibers with more peripheral nuclei were increased in CTX-injected WT mice by overloading. The muscle satellite cells, neuromuscular junctions (NMJ), and myonuclei were stained. Most of the properties, such as number of myonuclei and satellite cells, size of NMJ, and fiber length, were not influenced by mechanical overloading in all mice. Approximately 0.6% branched fibers were seen in the intact soleus of mdx mice, although these fibers were not detected in WT mice. However, the percentage of these fibers was increased by overloading especially in mdx mice (~50% vs. ~2.5% in WT). In CTX-injected WT mice, these fibers were ~15% with or without overloading. The fiber cross sectional area in normal WT, but not in mdx and CTX-injected WT mice, was increased by overloading (p<0.05). These results suggested that the functional overload induced muscle damage in mdx mice, but promoted the regeneration in CTX-injected WT mice.

  7. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    PubMed

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. PMID:24469912

  8. Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis

    PubMed Central

    2014-01-01

    Background Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy. Methods mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice. Results mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells. Conclusions These results suggest that andrographolide could be used to improve quality of life in individuals with DMD. PMID:24655808

  9. Fructose Promotes Uptake and Activity of Oligonucleotides With Different Chemistries in a Context-dependent Manner in mdx Mice.

    PubMed

    Cao, Limin; Han, Gang; Lin, Caorui; Gu, Ben; Gao, Xianjun; Moulton, Hong M; Seow, Yiqi; Yin, HaiFang

    2016-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise in correcting frame-disrupting mutations in the DMD gene for Duchenne muscular dystrophy. However, insufficient systemic delivery limits clinical adoption. Previously, we showed that a glucose/fructose mixture augmented AO delivery to muscle in mdx mice. Here, we evaluated if fructose alone could enhance the activities of AOs with different chemistries in mdx mice. The results demonstrated that fructose improved the potency of AOs tested with the greatest effect on phosphorodiamidate morpholino oligomer (PMO), resulted in a 4.25-fold increase in the number of dystrophin-positive fibres, compared to PMO in saline in mdx mice. Systemic injection of lissamine-labeled PMO with fructose at 25 mg/kg led to increased uptake and elevated dystrophin expression in peripheral muscles, compared to PMO in saline, suggesting that fructose potentiates PMO by enhancing uptake. Repeated intravenous administration of PMO in fructose at 50 mg/kg/week for 3 weeks and 50 mg/kg/month for 5 months restored up to 20% of wild-type dystrophin levels in skeletal muscles with improved functions without detectable toxicity, compared to untreated mdx controls. Collectively, we show that fructose can potentiate AOs of different chemistries in vivo although the effect diminished over repeated administration. PMID:27351681

  10. A Multidisciplinary Evaluation of the Effectiveness of Cyclosporine A in Dystrophic Mdx Mice

    PubMed Central

    De Luca, Annamaria; Nico, Beatrice; Liantonio, Antonella; Paola Didonna, Maria; Fraysse, Bodvael; Pierno, Sabata; Burdi, Rosa; Mangieri, Domenica; Rolland, Jean-François; Camerino, Claudia; Zallone, Alberta; Confalonieri, Paolo; Andreetta, Francesca; Arnoldi, Elisa; Courdier-Fruh, Isabelle; Magyar, Josef P.; Frigeri, Antonio; Pisoni, Michela; Svelto, Maria; Conte-Camerino, Diana

    2005-01-01

    Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy. PMID:15681831

  11. [WEAK COMBINED MAGNETIC FIELDS ADJUSTED TO THE PARAMETRIC RESONANCE FOR Ca2+ INTENSIFY DYSTROPHIN SYNTHESIS IN MDX MICE SKELETAL MUSCLES AFTER CELL THERAPY].

    PubMed

    Sokolova, A V; Sokolov, G V; Mikhailov, V M

    2016-01-01

    The mdx mice are an X-linked myopathic mutants, an animal model for human Duchenne muscular dystrophy (DMD). Mdx mice muscles are characterized by high level of striated muscle fibers (SMF) death followed by regeneration. As a result most SMFs of mdx mice have centrally located nuclei. The possibility of using stem cells therapy for the correction of DMD is actively being studied. One of the approaches to the usage of bone marrow stem cells for cellular therapy of DMD is the replacement of bone marrow after irradiation by X-rays. This method however does not give significant increase of dystrophin synthesis in mdx mice muscles fibers. We have tried to affect the mice after bone marrow transplantation by weak combined magnetic fields adjusted to the parametric resonance for Ca2+(Ca(2+)-MF) based on the data that the weak combined magnetic fields influence on tissues regeneration. We observed a significant increase in the proportion of dystrophin-positive SMFs in group of mdx mice radiation chimera 5 Gy and 3 Gy which was additionally exposed in Ca(2+)-MF in comparison with the control mdx mice and the group of mdx mice radiation chimera 5 Gy and 3 Gy which was kept in terrestrial magnetic field 2 months after chimera preparation--up to 15.8 and 18.3%, respectively. Also, there was an accumulation of SMFs without central nuclei. These data indicate a significanly increased efficacy of cell therapy in the case of additional exposition in Ca(2+)-MF. Thus, the efficiency of bone marrow transplantation mdx mice after both in doses 3 and 5 Gy was considerably enhanced by additional exposition to Ca(2+)-MF. Apparently, such magnetic field can intensify functioning of donor's nuclei which had been incorporated into muscle fibers. PMID:27228662

  12. Evolution of pathological changes in the gastrocnemius of the mdx mice correlate with utrophin and beta-dystroglycan expression.

    PubMed

    Roma, Josep; Munell, Francina; Fargas, Arnau; Roig, Manuel

    2004-11-01

    Utrophin can function in muscle as a substitute for dystrophin and its over-expression has been used successfully to ameliorate mdx muscle pathology. Despite of this fact, there are no detailed studies on the expression of endogenous skeletal muscle utrophin- and dystrophin-associated glycoproteins throughout the life span of mdx mice. We have monitored, sequentially, the expression of matrix metalloproteinase-9 (MMP-9), myosin heavy chain, utrophin and beta-dystroglycan, as well as the mRNA expression of utrophin and of structurally related proteins, in mdx and control mice. We found an inverse relationship between concentration of muscle utrophin and abundance of groups of degenerative-regenerative fibers and of MMP-9 expression. There was also temporal correlation between the decline of utrophin at 15 days of age and the onset of muscle necrosis. Conversely, reappearance of utrophin, with a peak around 2 months of age, was followed by a progressive decline of necrosis. A lineal correlation between utrophin and beta-dystroglycan levels, not seen in controls, indicates that improvement of mdx is due to utrophin binding to dystrophin-associated glycoproteins. Utrophin and other structurally related protein transcripts were not up-regulated, suggesting a post-transcriptional regulation for utrophin in skeletal muscle. PMID:15365724

  13. Effect of the IL-1 Receptor Antagonist Kineret® on Disease Phenotype in mdx Mice

    PubMed Central

    Benny Klimek, Margaret E.; Sali, Arpana; Rayavarapu, Sree; Van der Meulen, Jack H.; Nagaraju, Kanneboyina

    2016-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by mutations in the dystrophin gene. The pathology of DMD manifests in patients with progressive muscle weakness, loss of ambulation and ultimately death. One of the characteristics of DMD is muscle inflammation, and dystrophin-deficient skeletal muscles produce higher levels of the pro-inflammatory cytokine interleukin 1β (IL-1β) in response to toll like receptor (TLR) stimulation compared to controls; therefore, blocking the IL-1β pathway could improve the disease phenotype in mdx mice, a mouse model of DMD. Kineret® or IL-1Ra is a recombinant IL-1 receptor antagonist approved by the FDA for treating rheumatoid arthritis. To determine the efficacy of IL-1Ra in a DMD model, we administered subcutaneous injections of saline control or IL-1Ra (25 mg/kg/day) to mdx mice daily for 45 days beginning at 5 weeks of age. Functional and histological parameters were measured at the conclusion of the study. IL-1Ra only partially inhibited this signaling pathway in this study; however, there were still interesting observations to be noted. For example, although not significantly changed, splenocytes from the IL-1Ra-treated group secreted less IL-1β after LPS stimulation compared to control mice indicating a blunted response and incomplete inhibition of the pathway (37% decrease). In addition, normalized forelimb grip strength was significantly increased in IL-1Ra-treated mice. There were no changes in EDL muscle-specific force measurements, histological parameters, or motor coordination assessments in the dystrophic mice after IL-1Ra treatment. There was a significant 27% decrease in the movement time and total distance traveled by the IL-1Ra treated mice, correlating with previous studies examining effects of IL-1 on behavior. Our studies indicate partial blocking of IL-1β with IL-1Ra significantly altered only a few behavioral and strength related disease parameters; however, treatment with

  14. Effect of the IL-1 Receptor Antagonist Kineret® on Disease Phenotype in mdx Mice.

    PubMed

    Benny Klimek, Margaret E; Sali, Arpana; Rayavarapu, Sree; Van der Meulen, Jack H; Nagaraju, Kanneboyina

    2016-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by mutations in the dystrophin gene. The pathology of DMD manifests in patients with progressive muscle weakness, loss of ambulation and ultimately death. One of the characteristics of DMD is muscle inflammation, and dystrophin-deficient skeletal muscles produce higher levels of the pro-inflammatory cytokine interleukin 1β (IL-1β) in response to toll like receptor (TLR) stimulation compared to controls; therefore, blocking the IL-1β pathway could improve the disease phenotype in mdx mice, a mouse model of DMD. Kineret® or IL-1Ra is a recombinant IL-1 receptor antagonist approved by the FDA for treating rheumatoid arthritis. To determine the efficacy of IL-1Ra in a DMD model, we administered subcutaneous injections of saline control or IL-1Ra (25 mg/kg/day) to mdx mice daily for 45 days beginning at 5 weeks of age. Functional and histological parameters were measured at the conclusion of the study. IL-1Ra only partially inhibited this signaling pathway in this study; however, there were still interesting observations to be noted. For example, although not significantly changed, splenocytes from the IL-1Ra-treated group secreted less IL-1β after LPS stimulation compared to control mice indicating a blunted response and incomplete inhibition of the pathway (37% decrease). In addition, normalized forelimb grip strength was significantly increased in IL-1Ra-treated mice. There were no changes in EDL muscle-specific force measurements, histological parameters, or motor coordination assessments in the dystrophic mice after IL-1Ra treatment. There was a significant 27% decrease in the movement time and total distance traveled by the IL-1Ra treated mice, correlating with previous studies examining effects of IL-1 on behavior. Our studies indicate partial blocking of IL-1β with IL-1Ra significantly altered only a few behavioral and strength related disease parameters; however, treatment with

  15. Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice.

    PubMed

    Watchko, Jon; O'Day, Terry; Wang, Bing; Zhou, Liqiao; Tang, Ying; Li, Juan; Xiao, Xiao

    2002-08-10

    Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, afflicting 1 of every 3500 males. Patients with DMD experience progressive muscle degeneration and weakness and succumb to respiratory or cardiac failure by their early twenties. No treatment is currently available for DMD. Mutations in the dystrophin gene result in lack of a functional dystrophin protein in striated muscle, which induces instability in the muscle cell membrane leading to persistent muscle injury after contraction. We have previously created novel minidystrophin genes and demonstrated that adeno-associated virus (AAV)-mediated intramuscular delivery of the minigenes effectively ameliorated mdx dystrophic histopathology and led to normal cell membrane integrity for more than 1 year. In this paper, we investigated whether AAV-minidystrophin could also improve mdx muscle contractile function. Two-month-old adult male mdx mice, with established muscular dystrophy, were given a single-dose injection of an AAV-minidystrophin vector in the tibialis anterior (TA) muscle of one leg, with the untreated contralateral leg used as a control. The treated TA muscle showed both (1) a significant increase in isometric force generation and (2) a significant increase in resistance to lengthening activation-induced muscle force decrements. We conclude that AAV-minidystrophin gene treatment is effective in improving mdx muscle contractile function. PMID:12215266

  16. Preservation of Muscle Force in Mdx3cv Mice Correlates with Low-Level Expression of a Near Full-Length Dystrophin Protein

    PubMed Central

    Li, Dejia; Yue, Yongping; Duan, Dongsheng

    2008-01-01

    The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at ∼5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients. PMID:18385524

  17. Electrical Impedance Myography to Detect the Effects of Electrical Muscle Stimulation in Wild Type and Mdx Mice

    PubMed Central

    Li, Jia; Yim, Sung; Pacheck, Adam; Sanchez, Benjamin; Rutkove, Seward B.

    2016-01-01

    Objective Tools to better evaluate the impact of therapy on nerve and muscle disease are needed. Electrical impedance myography (EIM) is sensitive to neuromuscular disease progression as well as to therapeutic interventions including myostatin inhibition and antisense oligonucleotide-based treatments. Whether the technique identifies the impact of electrical muscle stimulation (EMS) is unknown. Methods Ten wild-type (wt) C57B6 mice and 10 dystrophin-deficient (mdx) mice underwent 2 weeks of 20 min/day EMS on left gastrocnemius and sham stimulation on the right gastrocnemius. Multifrequency EIM data and limb girth were obtained before and at the conclusion of the protocol. Muscle weight, in situ force measurements, and muscle fiber histology were also assessed at the conclusion of the study. Results At the time of sacrifice, muscle weight was greater on the EMS-treated side than on the sham-stimulated side (p = 0.018 for wt and p = 0.007 for mdx). Similarly, in wt animals, EIM parameters changed significantly compared to baseline (resistance (p = 0.009), reactance (p = 0.0003) and phase (p = 0.002); these changes were due in part to reductions in the EIM values on the EMS-treated side and elevations on the sham-simulated side. Mdx animals showed analogous but non-significant changes (p = 0.083, p = 0.064, and p = 0.57 for resistance, reactance and phase, respectively). Maximal isometric force trended higher on the stimulated side in wt animals only (p = 0.06). Myofiber sizes in wt animals were also larger on the stimulated side than on the sham-stimulated side (p = 0.034); no significant difference was found in the mdx mice (p = 0.79). Conclusion EIM is sensitive to stimulation-induced muscle alterations in wt animals; similar trends are also present in mdx mice. The mechanisms by which these EIM changes develop, however, remains uncertain. Possible explanations include longer-term trophic effects and shorter-term osmotic effects. PMID:26986564

  18. Interleukin-15 Administration Improves Diaphragm Muscle Pathology and Function in Dystrophic mdx Mice

    PubMed Central

    Harcourt, Leah J.; Holmes, Anna Greer; Gregorevic, Paul; Schertzer, Jonathan D.; Stupka, Nicole; Plant, David R.; Lynch, Gordon S.

    2005-01-01

    Interleukin (IL)-15, a cytokine expressed in skeletal muscle, has been shown to have muscle anabolic effects in vitro and to slow muscle wasting in rats with cancer cachexia. Whether IL-15 has therapeutic potential for diseases such as Duchenne muscular dystrophy (DMD) is unknown. We examined whether IL-15 administration could ameliorate the dystrophic pathology in the diaphragm muscle of the mdx mouse, an animal model for DMD. Four weeks of IL-15 treatment improved diaphragm strength, a highly significant finding because respiratory function is a mortality predictor in DMD. Enhanced diaphragm function was associated with increased muscle fiber cross-sectional area and decreased collagen infiltration. IL-15 administration was not associated with changes in T-cell populations or alterations in specific components of the ubiquitin proteasome pathway. To determine the effects of IL-15 on myofiber regeneration, muscles of IL-15-treated and untreated wild-type mice were injured myotoxically, and their functional recovery was assessed. IL-15 had a mild anabolic effect, increasing fiber cross-sectional area after 2 and 6 days but not after 10 days. Our findings demonstrate that IL-15 administration improves the pathophysiology of dystrophic muscle and highlight a possible therapeutic role for IL-15 in the treatment of neuromuscular disorders especially in which muscle wasting is indicated. PMID:15793293

  19. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

    PubMed Central

    Jearawiriyapaisarn, Natee; Moulton, Hong M; Buckley, Brian; Roberts, Jennifer; Sazani, Peter; Fucharoen, Suthat; Iversen, Patrick L; Kole, Ryszard

    2009-01-01

    Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals. PMID:18545222

  20. Protein-DNA array-based identification of transcription factor activities differentially regulated in skeletal muscle of normal and dystrophin-deficient mdx mice.

    PubMed

    Dogra, Charu; Srivastava, Daya Shankar; Kumar, Ashok

    2008-05-01

    Inactivation of dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and mdx mice. However, the underpinning mechanisms, which govern the pathogenesis of dystrophin-deficient skeletal muscle, remain poorly understood. We have previously reported activation of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-kappaB), and phosphatidyl-inositol 3-kinase/Akt (PI3K/Akt) signaling pathways in diaphragm muscle of mdx mice. In this study, using a protein-DNA array-based approach, we have investigated the activation of 345 transcription factors in diaphragm muscle of 6-week old normal and dystrophin-deficient mdx mice. Our data demonstrate increased activation of a number nuclear transcription factors including AP1, HFH-3, PPARalpha, c.myb BP, ETF, Fra-1/JUN, kBF-A, N-rasBP, lactoferrin BP, Myb(2), EBP40_45, EKLF(1), p53(2), TFEB, Myc-Max; c-Rel; E2, ISRE; NF-kB; Stat1 p84/p91, Antioxidant RE, EVI-1, Stat3, AP3, p53, Stat4, AP4, HFH-1, FAST-1, Pax-5, and Beta-RE in the diaphragm muscle of mdx mice compared to corresponding normal mice. The level of activation for p53 was highest among all the transcription factors studied. Furthermore, higher activation of p53 in diaphragm muscle of mdx mice was associated with its increased phosphorylation and nuclear translocation. Collectively, our data suggest that the primary deficiency of dystrophin leads to the aberrant activation of nuclear transcription factors which might further contribute to muscle pathogenesis in mdx mice. PMID:18278580

  1. Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy

    PubMed Central

    Giordano, Christian; Mojumdar, Kamalika; Liang, Feng; Lemaire, Christian; Li, Tong; Richardson, John; Divangahi, Maziar; Qureshi, Salman; Petrof, Basil J.

    2015-01-01

    Toll-like receptor 4 (TLR4) recognizes specific structural motifs associated with microbial pathogens and also responds to certain endogenous host molecules associated with tissue damage. In Duchenne muscular dystrophy (DMD), inflammation plays an important role in determining the ultimate fate of dystrophic muscle fibers. In this study, we used TLR4-deficient dystrophic mdx mice to assess the role of TLR4 in the pathogenesis of DMD. TLR4 expression was increased and showed enhanced activation following agonist stimulation in mdx diaphragm muscle. Genetic ablation of TLR4 led to significantly increased muscle force generation in dystrophic diaphragm muscle, which was associated with improved histopathology including decreased fibrosis, as well as reduced pro-inflammatory gene expression and macrophage infiltration. TLR4 ablation in mdx mice also altered the phenotype of muscle macrophages by inducing a shift toward a more anti-inflammatory (iNOSneg CD206pos) profile. In vitro experiments confirmed that lack of TLR4 is sufficient to influence macrophage activation status in response to classical polarizing stimuli such as IFN-gamma and IL-4. Finally, treatment of dystrophic mice with glycyrrhizin, an inhibitor of the endogenous TLR4 ligand, high mobility group box (HMGB1), also pointed to involvement of the HMGB1–TLR4 axis in promoting dystrophic diaphragm pathology. Taken together, our findings reveal TLR4 and the innate immune system as important players in the pathophysiology of DMD. Accordingly, targeting either TLR4 or its endogenous ligands may provide a new therapeutic strategy to slow disease progression. PMID:25552658

  2. Comparative study of myocytes from normal and mdx mice iPS cells.

    PubMed

    Chen, Fei; Cao, Jiqing; Liu, Qiang; Qin, Jie; Kong, Jie; Wang, Yanyun; Li, Yaqin; Geng, Jia; Li, Qiuling; Yang, Liqing; Xiang, Andy Peng; Zhang, Cheng

    2012-02-01

    Recently, induced pluripotent stem cells (iPS cells) have been derived from various techniques and show great potential for therapy of human diseases. Furthermore, the iPS technique can be used to provide cell models to explore pathological mechanisms of many human diseases in vitro, such as Duchenne muscular dystrophy (DMD), which is a severe recessive X-linked form of muscular dystrophy without effective treatment. In this study, we try to determine whether there are different characteristics of myocytes from mdx iPS cells and C57BL/10 iPS cells. Our results showed that both of mdx and C57BL/10 cells could be induced into iPS cells in vitro, whereas colony-forming ability of mdx iPS cells was much weaker than that of C57BL/10 iPS cells. Meanwhile, mdx iPS cells could be induced to differentiate into myocytes, whereas their differentiation efficiency was much lower than that of C57BL/10 iPS cells. And, the number of apoptotic cells in differentiated myocytes from mdx iPS cells was significantly higher than that from C57BL/10 iPS cells. More importantly, treatment of a pan-caspase inhibitor (Z-VAD) produced a significant decrease in apoptotic cells. This study might add some insight to the biology study of dystrophin gene. PMID:21976068

  3. Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice

    PubMed Central

    Froehner, Stanley C.; Reed, Sarah M.; Anderson, Kendra N.; Huang, Paul L.; Percival, Justin M.

    2015-01-01

    Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSβ, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSβ and use mdx mice engineered to lack nNOSμ and nNOSβ to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSβ was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSμ and nNOSβ is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSβ as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies. PMID:25214536

  4. Effective Dystrophin Restoration by a Novel Muscle-Homing Peptide–Morpholino Conjugate in Dystrophin-Deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Zhao, Jingwen; Han, Gang; Zhang, Yajie; Dong, Xue; Cao, Limin; Wang, Qingsong; Moulton, Hong M; Yin, HaiFang

    2014-01-01

    Antisense oligonucleotide (AO)–mediated splice correction therapy for Duchenne muscular dystrophy has shown huge promise from recent phase 2b clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide–AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to phosphorodiamidate morpholino oligomers, ~25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas <2% in corresponding tissues treated with either muscle-specific peptide–phosphorodiamidate morpholino oligomer or unmodified phosphorodiamidate morpholino oligomer under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-phosphorodiamidate morpholino oligomer can be an alternative option to current AOs. PMID:24732757

  5. Sulforaphane mitigates muscle fibrosis in mdx mice via Nrf2-mediated inhibition of TGF-β/Smad signaling.

    PubMed

    Sun, Chengcao; Li, Shujun; Li, Dejia

    2016-02-15

    Sulforaphane (SFN), an activator of NF-E2-related factor 2 (Nrf2), has been found to have an antifibrotic effect on liver and lung. However, its effects on dystrophic muscle fibrosis remain unknown. This work was undertaken to evaluate the effects of SFN-mediated activation of Nrf2 on dystrophic muscle fibrosis. Male mdx mice (age 3 mo) were treated with SFN by gavage (2 mg/kg body wt per day) for 3 mo. Experimental results demonstrated that SFN remarkably attenuated skeletal and cardiac muscle fibrosis as indicated by reduced Sirius Red staining and immunostaining of the extracellular matrix. Moreover, SFN significantly inhibited the transforming growth factor-β (TGF-β)/Smad signaling pathway and suppressed profibrogenic gene and protein expressions such as those of α-smooth muscle actin (α-SMA), fibronectin, collagen I, plasminogen activator inhibitor-1 (PAI-1), and tissue inhibitor metalloproteinase-1 (TIMP-1) in an Nrf2-dependent manner. Furthermore, SFN significantly decreased the expression of inflammatory cytokines CD45, TNF-α, and IL-6 in mdx mice. In conclusion, these results show that SFN can attenuate dystrophic muscle fibrosis by Nrf2-mediated inhibition of the TGF-β/Smad signaling pathway, which indicates that Nrf2 may represent a new target for dystrophic muscle fibrosis. PMID:26494449

  6. Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

    PubMed

    Jirka, Silvana M G; Heemskerk, Hans; Tanganyika-de Winter, Christa L; Muilwijk, Daan; Pang, Kar Him; de Visser, Peter C; Janson, Anneke; Karnaoukh, Tatyana G; Vermue, Rick; 't Hoen, Peter A C; van Deutekom, Judith C T; Aguilera, Begoña; Aartsma-Rus, Annemieke

    2014-02-01

    Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy. PMID:24320790

  7. Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice.

    PubMed

    Martin, Paul T; Xu, Rui; Rodino-Klapac, Louise R; Oglesbay, Elaine; Camboni, Marybeth; Montgomery, Chrystal L; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Sahenk, Zarife; Mendell, Jerry R; Janssen, Paul M L

    2009-03-01

    The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:beta1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcbeta1,4[NeuAc(orGc)alpha2, 3]Galbeta1,4GlcNAcbeta-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications. PMID:19109526

  8. Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction.

    PubMed

    Hyzewicz, Janek; Tanihata, Jun; Kuraoka, Mutsuki; Ito, Naoki; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

    2015-05-01

    High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein-protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs. PMID:25660994

  9. Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

    PubMed

    Gazzerro, Elisabetta; Baldassari, Simona; Assereto, Stefania; Fruscione, Floriana; Pistorio, Angela; Panicucci, Chiara; Volpi, Stefano; Perruzza, Lisa; Fiorillo, Chiara; Minetti, Carlo; Traggiai, Elisabetta; Grassi, Fabio; Bruno, Claudio

    2015-12-01

    Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-β and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy. PMID:26465071

  10. Whole Body Periodic Acceleration Is an Effective Therapy to Ameliorate Muscular Dystrophy in mdx Mice

    PubMed Central

    Altamirano, Francisco; Perez, Claudio F.; Liu, Min; Widrick, Jeffrey; Barton, Elisabeth R.; Allen, Paul D.; Adams, Jose A.; Lopez, Jose R.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca2+ and Na+ overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca2+ and Na+ overload, diminished abnormal sarcolemmal Ca2+ entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway. PMID:25181488

  11. Whole body periodic acceleration is an effective therapy to ameliorate muscular dystrophy in mdx mice.

    PubMed

    Altamirano, Francisco; Perez, Claudio F; Liu, Min; Widrick, Jeffrey; Barton, Elisabeth R; Allen, Paul D; Adams, Jose A; Lopez, Jose R

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca(2+) and Na(+) overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca(2+) and Na(+) overload, diminished abnormal sarcolemmal Ca(2+) entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway. PMID:25181488

  12. Lipid Peroxidation Inhibition Blunts Nuclear Factor-κB Activation, Reduces Skeletal Muscle Degeneration, and Enhances Muscle Function in mdx Mice

    PubMed Central

    Messina, Sonia; Altavilla, Domenica; Aguennouz, M’hammed; Seminara, Paolo; Minutoli, Letteria; Monici, Maria C.; Bitto, Alessandra; Mazzeo, Anna; Marini, Herbert; Squadrito, Francesco; Vita, Giuseppe

    2006-01-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease resulting from lack of the sarcolemmal protein dystrophin. However, the mechanism leading to the final disease status is not fully understood. Several lines of evidence suggest a role for nuclear factor (NF)-κB in muscle degeneration as well as regeneration in DMD patients and mdx mice. We investigated the effects of blocking NF-κB by inhibition of oxidative stress/lipid peroxidation on the dystrophic process in mdx mice. Five-week-old mdx mice received three times a week for 5 weeks either IRFI-042 (20 mg/kg), a strong antioxidant and lipid peroxidation inhibitor, or its vehicle. IRFI-042 treatment increased forelimb strength (+22%, P < 0.05) and strength normalized to weight (+23%, P < 0.05) and decreased fatigue (−45%, P < 0.05). It also reduced serum creatine kinase levels (P < 0.01) and reduced muscle-conjugated diene content and augmented muscle-reduced glutathione (P < 0.01). IRFI-042 blunted NF-κB DNA-binding activity and tumor necrosis factor-α expression in the dystrophic muscles (P < 0.01), reducing muscle necrosis (P < 0.01) and enhancing regeneration (P < 0.05). Our data suggest that oxidative stress/lipid peroxidation represents one of the mechanisms activating NF-κB and the consequent pathogenetic cascade in mdx muscles. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD. PMID:16507907

  13. The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

    PubMed Central

    Verhaart, Ingrid E C; van Vliet-van den Dool, Laura; Sipkens, Jessica A; de Kimpe, Sjef J; Kolfschoten, Ingrid G M; van Deutekom, Judith C T; Liefaard, Lia; Ridings, Jim E; Hood, Steve R; Aartsma-Rus, Annemieke

    2014-01-01

    Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD) to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides) will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks). Oligonucleotide half-life was longer in heart (~65 days) compared with that in skeletal muscle, liver, and kidney (~35 days). Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days). Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD. PMID:24549299

  14. D-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity.

    PubMed

    Reay, Daniel P; Bastacky, Sheldon I; Wack, Kathryn E; Stolz, Donna B; Robbins, Paul D; Clemens, Paula R

    2015-01-01

    In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using L-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring L-isoform peptide due to the longer persistence of the D-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of L- and D-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both L- or D-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the D-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice. PMID:26018805

  15. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice.

    PubMed

    Ragot, T; Vincent, N; Chafey, P; Vigne, E; Gilgenkrantz, H; Couton, D; Cartaud, J; Briand, P; Kaplan, J C; Perricaudet, M

    1993-02-18

    Duchenne progressive muscular dystrophy is a lethal and common X-linked genetic disease caused by the absence of dystrophin, a 427K protein encoded by a 14 kilobase transcript. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin. Adenovirus is an efficient vector for in vivo expression of various foreign genes. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle. PMID:8437625

  16. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    PubMed Central

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases. PMID:26366082

  17. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

    PubMed

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases. PMID:26366082

  18. Combination antisense treatment for destructive exon skipping of myostatin and open reading frame rescue of dystrophin in neonatal mdx mice

    PubMed Central

    Lu-Nguyen, Ngoc B.; Jarmin, Susan A.; Saleh, Amer F.; Popplewell, Linda; Gait, Michael J.; Dickson, George

    2015-01-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  19. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

    PubMed

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-08-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  20. Exercise restores decreased physical activity levels and increases markers of autophagy and oxidative capacity in myostatin/activin-blocked mdx mice.

    PubMed

    Hulmi, Juha J; Oliveira, Bernardo M; Silvennoinen, Mika; Hoogaars, Willem M H; Pasternack, Arja; Kainulainen, Heikki; Ritvos, Olli

    2013-07-15

    The importance of adequate levels of muscle size and function and physical activity is widely recognized. Myostatin/activin blocking increases skeletal muscle mass but may decrease muscle oxidative capacity and can thus be hypothesized to affect voluntary physical activity. Soluble activin receptor IIB (sActRIIB-Fc) was produced to block myostatin/activins. Modestly dystrophic mdx mice were injected with sActRIIB-Fc or PBS with or without voluntary wheel running exercise for 7 wk. Healthy mice served as controls. Running for 7 wk attenuated the sActRIIB-Fc-induced increase in body mass by decreasing fat mass. Running also enhanced/restored the markers of muscle oxidative capacity and autophagy in mdx mice to or above the levels of healthy mice. Voluntary running activity was decreased by sActRIIB-Fc during the first 3-4 wk correlating with increased body mass. Home cage physical activity of mice, quantified from the force plate signal, was decreased by sActRIIB-Fc the whole 7-wk treatment in sedentary mice. To understand what happens during the first weeks after sActRIIB-Fc administration, when mice are less active, healthy mice were injected with sActRIIB-Fc or PBS for 2 wk. During the sActRIIB-Fc-induced rapid 2-wk muscle growth period, oxidative capacity and autophagy were reduced, which may possibly explain the decreased running activity. These results show that increased muscle size and decreased markers of oxidative capacity and autophagy during the first weeks of myostatin/activin blocking are associated with decreased voluntary activity levels. Voluntary exercise in dystrophic mice enhances the markers of oxidative capacity and autophagy to or above the levels of healthy mice. PMID:23695214

  1. Serotonin and Histamine Therapy Increases Tetanic Forces of Myoblasts, Reduces Muscle Injury, and Improves Grip Strength Performance of Dmd(mdx) Mice.

    PubMed

    Gurel, Volkan; Lins, Jeremy; Lambert, Kristyn; Lazauski, Joan; Spaulding, James; McMichael, John

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a recessive X-linked fatal disorder caused by a mutation in the dystrophin gene. Although several therapeutic approaches have been studied, none has led to substantial long-term effects in patients. The aim of this study was to test a serotonin and histamine (S&H) combination on human skeletal myoblasts and Dmd(mdx) mice for its effects on muscle strength and injury. Normal human bioartificial muscles (BAMs) were treated, and muscle tetanic forces and muscle injury tests were performed using the MyoForce Analysis System. Dmd(mdx) mice, the murine model of DMD, were administered serotonin, histamine, or S&H combination twice daily for 6 weeks, and functional performance tests were conducted once a week. The S&H combination treatment caused significant increases in tetanic forces at all time points and concentrations tested as compared to the saline controls. Dose response of the BAMs to the treatment demonstrated a significant increase in force generation at all concentrations compared to the controls after 3 to 4 days of drug treatment. The highest 3 concentrations had a significant effect on lowering contractile-induced injury as measured by a reduction in the release of adenylate kinase. Histamine-only and S&H treatments improved grip strength of Dmd(mdx) mice, whereas serotonin-only treatment resulted in no significant improvement in muscle strength. The results of this study indicate that S&H therapy might be a promising new strategy for muscular dystrophies and that the mechanism should be further investigated. PMID:26740813

  2. Comparative study of inorganic elements determined in whole blood from Dmd(mdx)/J mice strain by EDXRF and NAA analytical techniques.

    PubMed

    Redígolo, M M; Sato, I M; Metairon, S; Zamboni, C B

    2016-04-01

    Several diseases can be diagnosed observing the variation of specific elements concentration in body fluids. In this study the concentration of inorganic elements in blood samples of dystrophic (Dmd(mdx)/J) and C57BL/6J (control group) mice strain were determined. The results obtained from Energy Dispersive X-ray Fluorescence (EDXRF) were compared with Neutron Activation Analysis (NAA) technique. Both analytical techniques showed to be appropriate and complementary offering a new contribution for veterinary medicine as well as detailed knowledge of this pathology. PMID:26826356

  3. Cardiac expression of a mini-dystrophin that normalizes skeletal muscle force only partially restores heart function in aged Mdx mice.

    PubMed

    Bostick, Brian; Yue, Yongping; Long, Chun; Marschalk, Nate; Fine, Deborah M; Chen, Jing; Duan, Dongsheng

    2009-02-01

    Duchenne muscular dystrophy (DMD) affects both skeletal and cardiac muscle. It is currently unclear whether the strategies developed for skeletal muscle can ameliorate cardiomyopathy. Synthetic mini-/micro-dystrophin genes have yielded impressive skeletal muscle protection in animal models. The 6-kb DeltaH2-R19 minigene is particularly promising because it completely restores skeletal muscle force to wild-type levels. Here, we examined whether expressing this minigene in the heart, but not skeletal muscle, could normalize cardiac function in the mdx model of DMD cardiomyopathy. Transgenic mdx mice were generated to express the DeltaH2-R19 minigene under the control of the alpha-myosin heavy-chain promoter. Heart structure and function were examined in adult and very old mice. The DeltaH2-R19 minigene enhanced cardiomyocyte sarcolemmal strength and prevented myocardial fibrosis. It also restored the dobutamine response and enhanced treadmill performance. Surprisingly, heart-restricted DeltaH2-R19 minigene expression did not completely normalize electrocardiogram and hemodynamic abnormalities. Overall, systolic function and ejection fraction were restored to normal levels but stroke volume and cardiac output remained suboptimal. Our results demonstrate that the skeletal muscle-proven DeltaH2-R19 minigene can correct cardiac histopathology but cannot fully normalize heart function. Novel strategies must be developed to completely restore heart function in DMD. PMID:19066599

  4. Poly(ester amine) Composed of Polyethylenimine and Pluronic Enhance Delivery of Antisense Oligonucleotides In Vitro and in Dystrophic mdx Mice.

    PubMed

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Bollinger, Lauren E; Lu, Peijuan; Lu, Qilong

    2016-01-01

    A series of poly(esteramine)s (PEAs) constructed from low molecular weight polyethyleneimine (LPEI) and Pluronic were evaluated for the delivery of antisense oligonuclotides (AOs), 2'-O-methyl phosphorothioate RNA (2'-OMePS) and phosphorodiamidate morpholino oligomer (PMO) in cell culture and dystrophic mdx mice. Improved exon-skipping efficiency of both 2'-OMePS and PMO was observed in the C2C12E50 cell line with all PEA polymers compared with PEI 25k or LF-2k. The degree of efficiency was found in the order of PEA 01, PEA 04 > PEA 05 > others. The in vivo study in mdx mice demonstrated enhanced exon-skipping of 2'-OMePS with the order of PEA 06 > PEA 04, PEA 07 > PEA 03 > PEA 01 > others, and much higher than PEI 25k formulated 2'-OMePS. Exon-skipping efficiency of PMO in formulation with the PEAs were significantly enhanced in the order of PEA 02 > PEA 10 > PEA 01, PEA 03 > PEA 05, PEA 07, PEA 08 > others, with PEA 02 reaching fourfold of Endo-porter formulated PMO. PEAs improve PMO delivery more effectively than 2'-OMePS delivery in vivo, and the systemic delivery evaluation further highlight the efficiency of PEA for PMO delivery in all skeletal muscle. The results suggest that the flexibility of PEA polymers could be explored for delivery of different AO chemistries, especially for antisense therapy. PMID:27483024

  5. GABA(A) receptor expression and inhibitory post-synaptic currents in cerebellar Purkinje cells in dystrophin-deficient mdx mice.

    PubMed

    Kueh, S L L; Head, S I; Morley, J W

    2008-02-01

    1. Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease and arises as a consequence of an absence or disruption of the protein dystrophin. In addition to wasting of the skeletal musculature, boys with DMD have a significant degree of cognitive impairment. 2. We show here that there is no difference between littermate control and mdx mice (a murine model of DMD) in the overall expression of the GABA(A) receptor a1-subunit, supporting the suggestion that it is the clustering at the synapse that is affected and not the expression of the GABA(A) receptor protein. 3. We report a significant reduction in both the frequency and amplitude of spontaneous inhibitory post-synaptic currents in cerebellar Purkinje cells of mdx mice compared with littermate controls, consistent with the reported reduction in the number and size of GABA(A) receptor clusters immunoreactive for a1- and a2-subunits at the post-synaptic densities. 4. These results may explain some of the behavioural problems and cognitive impairment reported in DMD. PMID:17941889

  6. MicroRNAs modulated by local mIGF-1 expression in mdx dystrophic mice

    PubMed Central

    Pelosi, Laura; Coggi, Angela; Forcina, Laura; Musarò, Antonio

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a X-linked genetic disease in which the absence of dystrophin leads to progressive lethal skeletal muscle degeneration. It has been demonstrated that among genes which are important for proper muscle development and function, micro-RNAs (miRNAs) play a crucial role. Moreover, altered levels of miRNAs were found in several muscular disorders, including DMD. A specific group of miRNAs, whose expression depends on dystrophin levels and whose deregulation explains several DMD pathogenetic traits, has been identified. Here, we addressed whether the anabolic activity of mIGF-1 on dystrophic muscle is associated with modulation of microRNAs expression. We demonstrated that some microRNAs are strictly linked to the dystrophin expression and are not modulated by mIGF-1 expression. In contrast, local expression of mIGF-1 promotes the modulation of other microRNAs, such as miR-206 and miR-24, along with the modulation of muscle specific genes, which are associated with maturation of regenerating fibers and with the stabilization of the differentiated muscle phenotype. These data suggest that mIGF-1, modifying the expression of some of the active players of muscle homeostasis, is able, even in absence of dystrophin expression, to activate circuitries that confer robustness to dystrophic muscle. PMID:25999854

  7. Evaluation of Tris[2-(Acryloyloxy)Ethyl]Isocyanurate Cross-Linked Polyethylenimine as Antisense Morpholino Oligomer Delivery Vehicle in Cell Culture and Dystrophic mdx Mice

    PubMed Central

    Wu, Bo; Tucker, Jay D.; Lu, Peijuan; Cloer, Caryn

    2014-01-01

    Abstract Hyperbranched poly(ester amine)s (PEAs) based on tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) cross-linked low-molecular-weight polyethylenimine (Mw: 0.8k/1.2k/2.0k) have been evaluated for delivering antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in vivo in the dystrophic mdx mouse. The results show that the PEAs constructed with polyethylenimine (PEI) 2.0k (C series) improved PMO delivery more efficiently than those constructed with PEI 0.8k (A series) or 1.2k (B series) in a GFP reporter-based C2C12 mouse myoblast culture system. The highest efficiency of exon-skipping in vitro with the PMO oligonucleotide targeting human dystrophin exon 50 was obtained when the PEA C12 [TAEI-PEI 2.0k (1:2)] was used. Nearly all of the PEAs improved dystrophin expression in mdx mice by local injection with a 2–4-fold increase when compared with PMO alone. Improved transfection efficiency and lower toxicity indicate the potential of the biodegradable PEA polymers as safe and efficient PMO delivery vectors for in vivo applications. PMID:24405395

  8. Evaluation of Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked polyethylenimine as antisense morpholino oligomer delivery vehicle in cell culture and dystrophic mdx mice.

    PubMed

    Wang, Mingxing; Wu, Bo; Tucker, Jay D; Lu, Peijuan; Cloer, Caryn; Lu, Qi Long

    2014-05-01

    Hyperbranched poly(ester amine)s (PEAs) based on tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) cross-linked low-molecular-weight polyethylenimine (Mw: 0.8k/1.2k/2.0k) have been evaluated for delivering antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in vivo in the dystrophic mdx mouse. The results show that the PEAs constructed with polyethylenimine (PEI) 2.0k (C series) improved PMO delivery more efficiently than those constructed with PEI 0.8k (A series) or 1.2k (B series) in a GFP reporter-based C2C12 mouse myoblast culture system. The highest efficiency of exon-skipping in vitro with the PMO oligonucleotide targeting human dystrophin exon 50 was obtained when the PEA C12 [TAEI-PEI 2.0k (1:2)] was used. Nearly all of the PEAs improved dystrophin expression in mdx mice by local injection with a 2-4-fold increase when compared with PMO alone. Improved transfection efficiency and lower toxicity indicate the potential of the biodegradable PEA polymers as safe and efficient PMO delivery vectors for in vivo applications. PMID:24405395

  9. Physiological Characterization of Muscle Strength With Variable Levels of Dystrophin Restoration in mdx Mice Following Local Antisense Therapy

    PubMed Central

    Sharp, Paul S; Bye-a-Jee, Hema; Wells, Dominic J

    2011-01-01

    Antisense-induced exon skipping can restore the open reading frame, and thus correct the dystrophin deficiency that causes Duchenne muscular dystrophy (DMD), a lethal muscle wasting condition. Successful proof-of-principle in preclinical models has led to human clinical trials. However, it is still not known what percentage of dystrophin-positive fibers and what level of expression is necessary for functional improvement. This study directly address these key questions in the mdx mouse model of DMD. To achieve a significant variation in dystrophin expression, we locally administered into tibialis anterior muscles various doses of a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin. We found a highly significant correlation between the number of dystrophin-positive fibers and resistance to contraction-induced injury, with a minimum of 20% of dystrophin-positive fibers required for meaningful improvement. Furthermore, our results also indicate that a relatively low level of dystrophin expression in muscle fibers may have significant clinical benefits. In contrast, improvements in muscle force were not correlated with either the number of positive fibers or total dystrophin levels, which highlight the need to conduct appropriate functional assessments in preclinical testing using the mdx mouse. PMID:20924363

  10. Sulforaphane Attenuates Muscle Inflammation in Dystrophin-deficient mdx Mice via NF-E2-related Factor 2 (Nrf2)-mediated Inhibition of NF-κB Signaling Pathway.

    PubMed

    Sun, Cheng-Cao; Li, Shu-Jun; Yang, Cui-Li; Xue, Rui-Lin; Xi, Yong-Yong; Wang, Liang; Zhao, Qian-Long; Li, De-Jia

    2015-07-17

    Inflammation is widely distributed in patients with Duchenne muscular dystrophy and ultimately leads to progressive deterioration of muscle function with chronic muscle damage, oxidative stress, and reduced oxidative capacity. NF-E2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzyme heme oxygenase-1 and inhibition of the NF-κB signaling pathway. However, the role of Nrf2 in the inflammation of dystrophic muscle remains unknown. To determine whether Nrf2 may counteract inflammation in dystrophic muscle, we treated 4-week-old male mdx mice with the Nrf2 activator sulforaphane (SFN) by gavage (2 mg/kg of body weight/day) for 4 weeks. The experimental results demonstrated that SFN treatment increased the expression of muscle phase II enzyme heme oxygenase-1 in an Nrf2-dependent manner. Inflammation in mice was reduced by SFN treatment as indicated by decreased infiltration of immune cells and expression of the inflammatory cytokine CD45 and proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the skeletal muscles of mdx mice. In addition, SFN treatment also decreased the expression of NF-κB(p65) and phosphorylated IκB kinase-α as well as increased inhibitor of κB-α expression in mdx mice in an Nrf2-dependent manner. Collectively, these results show that SFN-induced Nrf2 can alleviate muscle inflammation in mdx mice by inhibiting the NF-κB signaling pathway. PMID:26013831

  11. Electrical Stimuli Are Anti-Apoptotic in Skeletal Muscle via Extracellular ATP. Alteration of This Signal in Mdx Mice Is a Likely Cause of Dystrophy

    PubMed Central

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. PMID:24282497

  12. Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy.

    PubMed

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. PMID:24282497

  13. Differential expression of utrophin-A and -B promoters in the central nervous system (CNS) of normal and dystrophic mdx mice.

    PubMed

    Baby, Santhosh M; Bogdanovich, Sasha; Willmann, Gabriel; Basu, Utpal; Lozynska, Olga; Khurana, Tejvir S

    2010-03-01

    Utrophin (Utrn) is the autosomal homolog of dystrophin, the Duchene Muscular Dystrophy (DMD) locus product and of therapeutic interest, as its overexpression can compensate dystrophin's absence. Utrn is transcribed by Utrn-A and -B promoters with mRNAs differing at their 5' ends. However, previous central nervous system (CNS) studies used C-terminal antibodies recognizing both isoforms. As this distinction may impact upregulation strategies, we generated Utrn-A and -B promoter-specific antibodies, Taqman Polymerase chain reaction (PCR)-based absolute copy number assays, and luciferase-reporter constructs to study CNS of normal and dystrophic mdx mice. Differential expression of Utrn-A and -B was noted in microdissected and capillary-enriched fractions. At the protein level, Utrn-B was predominantly expressed in vasculature and ependymal lining, whereas Utrn-A was expressed in neurons, astrocytes, choroid plexus and pia mater. mRNA quantification demonstrated matching patterns of differential expression; however, transcription-translation mismatch was noted for Utrn-B in caudal brain regions. Utrn-A and Utrn-B proteins were significantly upregulated in olfactory bulb and cerebellum of mdx brain. Differential promoter activity, mRNA and protein expressions were studied in cultured C2C12, bEnd3, neurons and astrocytes. Promoter activity ranking for Utrn-A and -B was neurons > astrocytes > C2C12 > bEnd3 and bEnd3 > astrocytes > neurons > C2C12, respectively. Our results identify promoter usage patterns for therapeutic targeting and define promoter-specific differential distribution of Utrn isoforms in normal and dystrophic CNS. PMID:19486009

  14. Bubble liposomes and ultrasound exposure improve localized morpholino oligomer delivery into the skeletal muscles of dystrophic mdx mice.

    PubMed

    Negishi, Yoichi; Ishii, Yuko; Shiono, Hitomi; Akiyama, Saki; Sekine, Shoko; Kojima, Takuo; Mayama, Sayaka; Kikuchi, Taiki; Hamano, Nobuhito; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo; Aramaki, Yukihiko

    2014-03-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder that is caused by mutations in the DMD gene that lead to an absence of functional protein. The mdx dystrophic mouse contains a nonsense mutation in exon 23 of the dystrophin gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression. However, an efficient PMO delivery method is needed to improve treatment strategies for DMD. We previously developed polyethylene glycol (PEG)-modified liposomes (Bubble liposomes) that entrap ultrasound contrast gas and demonstrated that the combination of Bubble liposomes with ultrasound exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of Bubble liposomes as a PMO delivery tool, we tested the potency of the Bubble liposomes combined with ultrasound exposure to boost the delivery of PMO and increase the skipping of the mutated exon in the mdx mouse. The results indicated that the combination of Bubble liposomes and ultrasound exposure increased the uptake of the PMO targeting a nonsense mutation in exon 23 of the dystrophin gene and consequently increased the PMO-mediated exon-skipping efficiency compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency indicated the potential of the combination of Bubble liposomes with ultrasound exposure to enhance PMO delivery for treating DMD. Thus, this ultrasound-mediated Bubble liposome technique may provide an effective, noninvasive, nonviral method for PMO therapy for DMD muscle as well as for other muscular dystrophies. PMID:24433046

  15. Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity.

    PubMed

    Eghtesad, S; Zheng, H; Nakai, H; Epperly, M W; Clemens, P R

    2010-09-01

    Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle. PMID:20827278

  16. Multivariate Data EXplorer (MDX)

    SciTech Connect

    Steed, Chad Allen

    2012-08-01

    The MDX toolkit facilitates exploratory data analysis and visualization of multivariate datasets. MDX provides and interactive graphical user interface to load, explore, and modify multivariate datasets stored in tabular forms. MDX uses an extended version of the parallel coordinates plot and scatterplots to represent the data. The user can perform rapid visual queries using mouse gestures in the visualization panels to select rows or columns of interest. The visualization panel provides coordinated multiple views whereby selections made in one plot are propagated to the other plots. Users can also export selected data or reconfigure the visualization panel to explore relationships between columns and rows in the data.

  17. Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45–55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice

    PubMed Central

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  18. Pluronic-PEI copolymers enhance exon-skipping of 2'-O-methyl phosphorothioate oligonucleotide in cell culture and dystrophic mdx mice.

    PubMed

    Wang, M; Wu, B; Lu, P; Tucker, J D; Milazi, S; Shah, S N; Lu, Q L

    2014-01-01

    A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2'-O-methyl phosphorothioate RNA (2'-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100-300 nm nanoparticles. Exon-skipping effect of 2'-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2'-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k-5kDa) and intermediate hydrophilic-lipophilic balance (7-23) of pluronics, enhanced exon-skipping of 2'-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases. PMID:24131982

  19. Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked low-molecular-weight polyethylenimine as gene delivery carriers in cell culture and dystrophic mdx mice.

    PubMed

    Wang, Mingxing; Tucker, Jay D; Lu, Peijuan; Wu, Bo; Cloer, Caryn; Lu, Qilong

    2012-04-18

    Hyperbranched poly(ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) and low-molecular-weight polyethylenimine (LPEI, M(w) 0.8k, 1.2k, and 2.0k) and evaluated in vitro and in vivo as gene carriers. PEAs effectively condensed plasmid DNA with particle sizes below 200 nm and surface charges between 11.5 and 33.5 mV under tested doses [at the ratios 2-10:1 of polymer/pDNA(w/w)]. The PEAs showed significantly lower cytotoxicities when compared with PEI 25k in two different cell lines. The PEAs (C series) composed of PEI 2k showed higher transgene expression compared to PEAs of PEI 0.8k (A series) or 1.2k (B series). Highest gene transfection efficiency in CHO, C2C12 myoblast, and human skeletal muscle (HSK) cell lines was obtained with TAEI/PEI-2K (C12) at a ratio of 1:2. Both C12, C14(TAEI/PEI-2K at a ratio of 1:4) demonstrated 5-8-fold higher gene expression as compared with PEI 25k in mdx mice in vivo through intramuscular administration. No obvious muscle damage was observed with these new polymers. Higher transfection efficiency and lower toxicity indicate the potential of the biodegradable PEAs as safe and efficient transgene delivery vectors. PMID:22443086

  20. Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

    PubMed

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45-55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45-55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45-55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45-55 region was induced, and the Western blot analysis exhibited the restoration of 5-27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  1. Long-term administration of the TNF blocking drug Remicade (cV1q) to mdx mice reduces skeletal and cardiac muscle fibrosis, but negatively impacts cardiac function.

    PubMed

    Ermolova, N V; Martinez, L; Vetrone, S A; Jordan, M C; Roos, K P; Sweeney, H L; Spencer, M J

    2014-07-01

    Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in the gene encoding dystrophin (DYS). Tumor necrosis factor (TNF) has been implicated in the pathogenesis since short-term treatment of mdx mice with TNF blocking drugs proved beneficial; however, it is not clear whether long-term treatment will also improve long-term outcomes of fibrosis and cardiac health. In this investigation, short and long-term dosing studies were carried out using the TNF blocking drug Remicade and a variety of outcome measures were assessed. Here we show no demonstrable benefit to muscle strength or morphology with 10mg/kg or 20mg/kg Remicade; however, 3mg/kg produced positive strength benefits. Remicade treatment correlated with reductions in myostatin mRNA in the heart, and concomitant reductions in cardiac and skeletal fibrosis. Surprisingly, although Remicade treated mdx hearts were less fibrotic, reductions in LV mass and ejection fraction were also observed, and these changes coincided with reductions in AKT phosphorylation on threonine 308. Thus, TNF blockade benefits mdx skeletal muscle strength and fibrosis, but negatively impacts AKT activation, leading to deleterious changes to dystrophic heart function. These studies uncover a previously unknown relationship between TNF blockade and alteration of muscle growth signaling pathways. PMID:24844454

  2. Exclusive skeletal muscle correction does not modulate dystrophic heart disease in the aged mdx model of Duchenne cardiomyopathy

    PubMed Central

    Wasala, Nalinda B.; Bostick, Brian; Yue, Yongping; Duan, Dongsheng

    2013-01-01

    Duchenne muscular dystrophy (DMD) is characterized by severe degeneration and necrosis of both skeletal and cardiac muscle. While many experimental therapies have shown great promise in treating skeletal muscle disease, an effective therapy for Duchenne cardiomyopathy remains a challenge in large animal models and human patients. The current views on cardiac consequences of skeletal muscle-centered therapy are controversial. Studies performed in young adult mdx mice (a mild DMD mouse model) have yielded opposing results. Since mdx mice do not develop dystrophic cardiomyopathy until ≥21 months of age, we reasoned that old mdx mice may represent a better model to assess the impact of skeletal muscle rescue on dystrophic heart disease. Here, we aged skeletal muscle-specific micro-dystrophin transgenic mdx mice to 23 months and examined the cardiac phenotype. As expected, transgenic mdx mice had minimal skeletal muscle disease and they also outperformed original mdx mice on treadmill running. On cardiac examination, the dystrophin-null heart of transgenic mdx mice displayed severe cardiomyopathy matching that of non-transgenic mdx mice. Specifically, both the strains showed similar heart fibrosis and cardiac function deterioration in systole and diastole. Cardiac output and ejection fraction were also equally compromised. Our results suggest that skeletal muscle rescue neither aggravates nor alleviates cardiomyopathy in aged mdx mice. These findings underscore the importance of treating both skeletal and cardiac muscles in DMD therapy. PMID:23459935

  3. Impaired response of mature adipocytes of diabetic mice to hypoxia

    SciTech Connect

    Hong, Seok Jong Jin, Da P.; Buck, Donald W.; Galiano, Robert D.; Mustoe, Thomas A.

    2011-10-01

    Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.

  4. Multivariate Data EXplorer (MDX)

    Energy Science and Technology Software Center (ESTSC)

    2012-08-01

    The MDX toolkit facilitates exploratory data analysis and visualization of multivariate datasets. MDX provides and interactive graphical user interface to load, explore, and modify multivariate datasets stored in tabular forms. MDX uses an extended version of the parallel coordinates plot and scatterplots to represent the data. The user can perform rapid visual queries using mouse gestures in the visualization panels to select rows or columns of interest. The visualization panel provides coordinated multiple views wherebymore » selections made in one plot are propagated to the other plots. Users can also export selected data or reconfigure the visualization panel to explore relationships between columns and rows in the data.« less

  5. Decrease in Prosaposin in the Dystrophic mdx Mouse Brain

    PubMed Central

    Gao, Hui-ling; Li, Cheng; Nabeka, Hiroaki; Shimokawa, Tetsuya; Kobayashi, Naoto; Saito, Shouichiro; Wang, Zhan-You; Cao, Ya-ming; Matsuda, Seiji

    2013-01-01

    Background Duchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities. Methodology/Principal Findings The distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells. Conclusions/Significance Low levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice. PMID:24244600

  6. X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD

    SciTech Connect

    Wakeford, S.; Watt, D.J.; Partridge, T.A. )

    1991-01-01

    The mdx mouse, although a genetic and biochemical homologue of human Duchenne muscular dystrophy (DMD), presents a comparatively mild histopathological and clinical phenotype. These differences are partially attributable to the greater efficacy of regeneration in the mdx mouse than in DMD muscle. To lessen this disparity, we have used a single dose of X-irradiation (16 Gy) to inhibit regeneration in one leg of mdx mice. The result is an almost complete block of muscle fiber regeneration leading to progressive loss of muscle fibers and their replacement by loose connective tissue. Surviving fibers are mainly peripherally nucleated and, surprisingly, of large diameter. Thus, X-irradiation converts mdx muscle to a model system in which the degenerative process can be studied in isolation from the complicating effect of myofiber regeneration. This system should be of use for testing methods of alleviating the myofiber degeneration which is common to mdx and DMD.

  7. Ventilatory chemosensory drive is blunted in the mdx mouse model of Duchenne Muscular Dystrophy (DMD).

    PubMed

    Mosqueira, Matias; Baby, Santhosh M; Lahiri, Sukhamay; Khurana, Tejvir S

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure. PMID:23922741

  8. Ventilatory Chemosensory Drive Is Blunted in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)

    PubMed Central

    Mosqueira, Matias; Baby, Santhosh M.; Khurana, Tejvir S.

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure. PMID:23922741

  9. Malformed mdx myofibers have normal cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+ signaling

    PubMed Central

    Ward, Christopher W.

    2009-01-01

    Skeletal muscle function is dependent on its highly regular structure. In studies of dystrophic (dy/dy) mice, the proportion of malformed myofibers decreases after prolonged whole muscle stimulation, suggesting that the malformed myofibers are more prone to injury. The aim of this study was to assess morphology and to measure excitation-contraction (EC) coupling (Ca2+ transients) and susceptibility to osmotic stress (Ca2+ sparks) of enzymatically isolated muscle fibers of the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles from young (2–3 mo) and old (8–9 mo) mdx and age-matched control mice (C57BL10). In young mdx EDL, 6% of the myofibers had visible malformations (i.e., interfiber splitting, branched ends, midfiber appendages). In contrast, 65% of myofibers in old mdx EDL contained visible malformations. In the mdx FDB, malformation occurred in only 5% of young myofibers and 11% of old myofibers. Age-matched control mice did not display the altered morphology of mdx muscles. The membrane-associated and cytoplasmic cytoskeletal structures appeared normal in the malformed mdx myofibers. In mdx FDBs with significantly branched ends, an assessment of global, electrically evoked Ca2+ signals (indo-1PE-AM) revealed an EC coupling deficit in myofibers with significant branching. Interestingly, peak amplitude of electrically evoked Ca2+ release in the branch of the bifurcated mdx myofiber was significantly decreased compared with the trunk of the same myofiber. No alteration in the basal myoplasmic Ca2+ concentration (i.e., indo ratio) was seen in malformed vs. normal mdx myofibers. Finally, osmotic stress induced the occurrence of Ca2+ sparks to a greater extent in the malformed portions of myofibers, which is consistent with deficits in EC coupling control. In summary, our data show that aging mdx myofibers develop morphological malformations. These malformations are not associated with gross disruptions in cytoskeletal or t

  10. Caspase-12 ablation preserves muscle function in the mdx mouse

    PubMed Central

    Moorwood, Catherine; Barton, Elisabeth R.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin. Several downstream consequences of dystrophin deficiency are triggers of endoplasmic reticulum (ER) stress, including loss of calcium homeostasis, hypoxia and oxidative stress. During ER stress, misfolded proteins accumulate in the ER lumen and the unfolded protein response (UPR) is triggered, leading to adaptation or apoptosis. We hypothesized that ER stress is heightened in dystrophic muscles and contributes to the pathology of DMD. We observed increases in the ER stress markers BiP and cleaved caspase-4 in DMD patient biopsies, compared with controls, and an increase in multiple UPR pathways in muscles of the dystrophin-deficient mdx mouse. We then crossed mdx mice with mice null for caspase-12, the murine equivalent of human caspase-4, which are resistant to ER stress. We found that deleting caspase-12 preserved mdx muscle function, resulting in a 75% recovery of both specific force generation and resistance to eccentric contractions. The compensatory hypertrophy normally found in mdx muscles was normalized in the absence of caspase-12; this was found to be due to decreased fibre sizes, and not to a fibre type shift or a decrease in fibrosis. Fibre central nucleation was not significantly altered in the absence of caspase-12, but muscle fibre degeneration found in the mdx mouse was reduced almost to wild-type levels. In conclusion, we have identified heightened ER stress and abnormal UPR signalling as novel contributors to the dystrophic phenotype. Caspase-4 is therefore a potential therapeutic target for DMD. PMID:24879640

  11. Defective regulation of energy metabolism in mdx-mouse skeletal muscles.

    PubMed

    Even, P C; Decrouy, A; Chinet, A

    1994-12-01

    Our previous finding of a reduced energy metabolism in slow- and fast-twitch skeletal muscle fibres from the murine model of Duchenne muscular dystrophy (the mdx mouse) led us to examine the importance of intracellular glucose availability for a normal energy turnover. To this end, basal and KCl-stimulated (20.9 mM total extracellular K+) rates of glucose uptake (GUP) and heat production were measured in isolated, glucose-incubated (5 mM) soleus and extensor digitorum longus muscles from mdx and control C57B1/10 mice, in the presence and in the absence of insulin (1.7 nM). Under all conditions and for both muscle types, glucose uptake values for mdx and control muscles were similar although heat production was lower in mdx muscles. The marked stimulation of GUP by insulin in both mdx and control muscles had only minor effects on heat production. In contrast, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose (5 mM) significantly decreased heat production in control muscles only, which attenuated, although did not suppress, the difference in basal heat production between mdx and control muscles. Stimulation of heat production by a short-chain fatty acid salt (octanoate, 2 mM) was significantly less marked in mdx than in control muscles. Increased cytoplasmic synthesis of CoA by addition of 5 mM pantothenate (vitamin B5) increased the thermogenic response to glucose more in mdx than in control muscles. We conclude that the low energy turnover in mdx-mouse muscle fibres is not due to a decrease of intracellular glucose availability, but rather to a decreased oxidative utilization of glucose and free fatty acids. We suggest that some enzyme complex of the tricarboxylic acid cycle or inefficiency of CoA transport in the mitochondria could be involved. PMID:7999003

  12. Diaphragm degeneration and cardiac structure in mdx mouse: potential clinical implications for Duchenne muscular dystrophy.

    PubMed

    Barbin, Isabel Cristina Chagas; Pereira, Juliano Alves; Bersan Rovere, Matheus; de Oliveira Moreira, Drielen; Marques, Maria Julia; Santo Neto, Humberto

    2016-05-01

    We examined the effects of exercise on diaphragm degeneration and cardiomyopathy in dystrophin-deficient mdx mice. Mdx mice (11 months of age) were exercised (swimming) for 2 months to worsen diaphragm degeneration. Control mdx mice were kept sedentary. Morphological evaluation demonstrated increased fibrosis in the diaphragm of exercised mdx mice (33.3 ± 6.0% area of fibrosis) compared with control mdx mice (20.9 ± 1.7% area of fibrosis). Increased (26%) activity of MMP-2, a marker of fibrosis, was detected in the diaphragms from exercised mdx mice. Morphological evaluation of the heart demonstrated a 45% increase in fibrosis in the right ventricle (8.3 ± 0.6% in sedentary vs. 12.0 ± 0.6% of fibrosis in exercised) and in the left ventricle (35% increase) in the exercised mdx mice. The density of inflammatory cells-degenerating cardiomyocytes increased 95% in the right ventricle (2.3 ± 0.6 in sedentary vs. 4.5 ± 0.8 in exercised) and 71% in the left ventricle (1.4 ± 0.6 sedentary vs. 2.4 ± 0.5 exercised). The levels of both active MMP-2 and the pro-fibrotic factor transforming growth factor beta were elevated in the hearts of exercised compared with sedentary mdx mice. The wall thickness to lumen diameter ratio of the pulmonary trunk was significantly increased in the exercised mdx mice (0.11 ± 0.04 in sedentary vs. 0.28 ± 0.12 in exercised), as was the thickness of the right ventricle wall, which suggests the occurrence of pulmonary hypertension in those animals. It is suggested that diaphragm degeneration is a main contributor to right ventricle dystrophic pathology. These findings may be relevant for future interventional studies for Duchenne muscular dystrophy-associated cardiomyopathy. PMID:26822140

  13. Beneficial cilostazol therapeutic effects in mdx dystrophic skeletal muscle.

    PubMed

    Hermes, Túlio de Almeida; Macedo, Aline Barbosa; Fogaça, Aline Reis; Moraes, Luis Henrique Rapucci; de Faria, Felipe Meira; Kido, Larissa Akemi; Cagnon, Valéria Helena Alves; Minatel, Elaine

    2016-02-01

    This study evaluated the possible protective effects of cilostazol against myonecrosis in dystrophic diaphragm muscle in vivo, focusing on oxidative stress, the inflammatory response and angiogenesis. Young mdx mice, the experimental animal for Duchenne muscular dystrophy, received cilostazol for 14 days. A second group of mdx mice and a control group of C57BL/10 mice received a saline solution. In the mdx mice, cilostazol treatment was associated with reduced loss of muscle strength (-34.4%), decreased myonecrosis, reduced creatine kinase levels (-63.3%) and muscle fibres stained for immunoglobulin G in dystrophic diaphragm muscle (-81.1%), and a reduced inflammatory response, with a decreased inflammatory area (-22%), macrophage infiltration (-44.9%) and nuclear factor-κB (-24%) and tumour necrosis factor-α (-48%) content in dystrophic diaphragm muscle. Furthermore, cilostazol decreased oxidative stress and attenuated reactive oxygen species production (-74%) and lipid peroxidation (-17%) in dystrophic diaphragm muscle, and promoted the up-regulation of angiogenesis, increasing the number of microvessels (15%). In conclusion, the present results show that cilostazol has beneficial effects in dystrophic muscle. More research into the potential of cilostazol as a novel therapeutic agent for the treatment of dystrophinopathies is required. PMID:26639107

  14. Major alteration of the pathological phenotype in gamma irradiated mdx soleus muscles

    SciTech Connect

    Weller, B.; Karpati, G.; Lehnert, S.; Carpenter, S. )

    1991-07-01

    Two thousand rads of gamma irradiation delivered to the lower legs of ten day old normal and x-chromosome linked muscular dystrophy (mdx) mice caused significant inhibition of tibial bone and soleus muscle fiber growth. In the irradiated mdx solei, there was a major loss of muscle fibers, lack of central nucleation, and some endomysial fibrosis. These features were caused by a failure of regeneration of muscle fibers due to impaired proliferative capacity of satellite cells. Gamma irradiation transforms the late pathological phenotype of mdx muscles, so that in one major aspect (muscle fiber loss) they resemble muscles in Duchenne muscular dystrophy. However, extensive endomysial fibrosis which is another characteristic feature of Duchenne muscular dystrophy did not develop. This experimental model could be useful for the functional investigation of possible beneficial effects of therapeutic interventions in mdx dystrophy.

  15. Early manifestation of alteration in cardiac function in dystrophin deficient mdx mouse using 3D CMR tagging

    PubMed Central

    Li, Wei; Liu, Wei; Zhong, Jia; Yu, Xin

    2009-01-01

    Background Duchenne muscular dystrophy (DMD) is caused by the absence of the cytoskeletal protein, dystrophin. In DMD patients, dilated cardiomyopathy leading to heart failure may occur during adolescence. However, early cardiac dysfunction is frequently undetected due to physical inactivity and generalized debilitation. The objective of this study is to determine the time course of cardiac functional alterations in mdx mouse, a mouse model of DMD, by evaluating regional ventricular function with CMR tagging. Methods In vivo myocardial function was evaluated by 3D CMR tagging in mdx mice at early (2 months), middle (7 months) and late (10 months) stages of disease development. Global cardiac function, regional myocardial wall strains, and ventricular torsion were quantified. Myocardial lesions were assessed with Masson's trichrome staining. Results Global contractile indexes were similar between mdx and C57BL/6 mice in each age group. Histology analysis showed that young mdx mice were free of myocardial lesions. Interstitial fibrosis was present in 7 month mdx mice, with further development into patches or transmural lesions at 10 months of age. As a result, 10 month mdx mice showed significantly reduced regional strain and torsion. However, young mdx mice showed an unexpected increase in regional strain and torsion, while 7 month mdx mice displayed similar regional ventricular function as the controls. Conclusion Despite normal global ventricular function, CMR tagging detected a biphasic change in myocardial wall strain and torsion, with an initial increase at young age followed by progressive decrease at older ages. These results suggest that CMR tagging can provide more sensitive measures of functional alterations than global functional indexes in dystrophin-related cardiomyopathies. PMID:19849858

  16. Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis.

    PubMed

    Cea, Luis A; Puebla, Carlos; Cisterna, Bruno A; Escamilla, Rosalba; Vargas, Aníbal A; Frank, Marina; Martínez-Montero, Paloma; Prior, Carmen; Molano, Jesús; Esteban-Rodríguez, Isabel; Pascual, Ignacio; Gallano, Pía; Lorenzo, Gustavo; Pian, Héctor; Barrio, Luis C; Willecke, Klaus; Sáez, Juan C

    2016-07-01

    Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca(2+) levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice was significantly better than that of control mdx Cx43(fl/fl)Cx45(fl/fl) mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD. PMID:26803842

  17. Metabolic profile of dystrophic mdx mouse muscles analyzed with in vitro magnetic resonance spectroscopy (MRS).

    PubMed

    Martins-Bach, Aurea B; Bloise, Antonio C; Vainzof, Mariz; Rahnamaye Rabbani, Said

    2012-10-01

    Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy characterized by progressive and irreversible degeneration of the muscles. The mdx mouse is the classical animal model for DMD, showing similar molecular and protein defects. The mdx mouse, however, does not show significant muscle weakness, and the diaphragm muscle is significantly more degenerated than skeletal muscles. In this work, (1)H magnetic resonance spectroscopy (MRS) was used to study the metabolic profile of quadriceps and diaphragm muscles from mdx and control mice. Using principal components analysis (PCA), the animals were separated into groups according to age and lineages. The classification was compared to histopathological analysis. Among the 24 metabolites identified from the nuclear MR spectra, only 19 were used by the PCA program for classification purposes. These can be important key biomarkers associated with the progression of degeneration in mdx muscles and with natural aging in control mice. Glutamate, glutamine, succinate, isoleucine, acetate, alanine and glycerol were increased in mdx samples as compared to control mice, in contrast to carnosine, taurine, glycine, methionine and creatine that were decreased. These results suggest that MRS associated with pattern recognition analysis can be a reliable tool to assess the degree of pathological and metabolic alterations in the dystrophic tissue, thereby affording the possibility of evaluation of beneficial effects of putative therapies. PMID:22673895

  18. Impaired dendritic cell maturation and increased T(H)2 responses in PIR-B(-/-) mice.

    PubMed

    Ujike, Azusa; Takeda, Kazuhiko; Nakamura, Akira; Ebihara, Shin; Akiyama, Kenichi; Takai, Toshiyuki

    2002-06-01

    Mice deficient for paired immunoglobulin (Ig)-like receptor B (PIR-B) show defective regulation of receptor-mediated activation in antigen-presenting cells. Older PIR-B(-/-) mice had an increased number of peritoneal B1 cells. Splenic PIR-B(-/-) B2 cells were constitutively activated and proliferated much more than those from wild-type mice upon B cell receptor ligation. T helper type 2 (T(H)2)-prone humoral responses were augmented in PIR-B(-/-) mice upon immunization with T-dependent antigens, including increased interleukin 4 and decreased interferon-gamma responses, as well as enhanced IgG1 and IgE production. Impaired maturation of dendritic cells (DCs), possibly due to perturbed intracellular signaling, was responsible for the skewed responses. Thus, PIR-B is critical for B cell suppression, DC maturation and for balancing T(H)1 and T(H)2 immune responses. PMID:12021780

  19. Biochemical and Functional Comparisons of mdx and Sgcg−/− Muscular Dystrophy Mouse Models

    PubMed Central

    Roberts, Nathan W.; Holley-Cuthrell, Jenan; Gonzalez-Vega, Magdalis; Mull, Aaron J.; Heydemann, Ahlke

    2015-01-01

    Mouse models have provided an essential platform to investigate facets of human diseases, from etiology, diagnosis, and prognosis, to potential treatments. Muscular dystrophy (MD) is the most common human genetic disease occurring in approximately 1 in 2500 births. The mdx mouse, which is dystrophin-deficient, has long been used to model this disease. However, this mouse strain displays a rather mild disease course compared to human patients. The mdx mice have been bred to additional genetically engineered mice to worsen the disease. Alternatively, other genes which cause human MD have been genetically disrupted in mice. We are now comparing disease progression from one of these alternative gene disruptions, the γ-sarcoglycan null mouse Sgcg−/− on the DBA2/J background, to the mdx mouse line. This paper aims to assess the time-course severity of the disease in the mouse models and determine which is best for MD research. The Sgcg−/− mice have a more severe phenotype than the mdx mice. Muscle function was assessed by plethysmography and echocardiography. Histologically the Sgcg−/− mice displayed increased fibrosis and variable fiber size. By quantitative Evan's blue dye uptake and hydroxyproline content two key disease determinants, membrane permeability and fibrosis respectively, were also proven worse in the Sgcg−/− mice. PMID:26064876

  20. The effect of respiratory muscle training with CO2 breathing on cellular adaptation of mdx mouse diaphragm

    PubMed Central

    Matécki, Stefan; Rivier, François; Hugon, Gerald; Koechlin, Christelle; Michel, Alain; Préfaut, Christian; Mornet, Dominique; Ramonatxo, Michèle

    2005-01-01

    The aim of our study was to investigate the cellular mechanisms induced by hypercapnic stimulation of ventilation, during 6 weeks/30 min per day, in 10 mdx and 8 C57BL10 mice (10G0.2 months old). Ten mdx and eight C57BL10 mice served as control group. This respiratory training increases in vitro maximal tetanic tension of the diaphragm only in mdx mice. Western blot analysis of diaphragm showed: (1) an over-expression of a-dystrobrevin in mdx and C57BL10 training group compared to control group (8100G710 versus 6100G520 and 2800G400 versus 2200G250 arbitrary units); (2) a decrease in utrophin expression only in mdx training group compared to control group (2100G320 versus 3100G125 arbitrary units). Daily respiratory muscle training in mdx mice, induces a beneficial effect on diaphragm strength, with an over-expression of a-dystrobrevin. Further studies are needed to determine if, in absence of dystrophin, the over-expression of a-dystrobrevin could be interpreted as a possible pathway to improve function of dystrophic muscle. PMID:15907290

  1. Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

    PubMed

    Wazen, Rima M; Viegas-Costa, Luiz C; Fouillen, Aurélien; Moffatt, Pierre; Adair-Kirk, Tracy L; Senior, Robert M; Nanci, Antonio

    2016-01-01

    The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation. PMID:26956061

  2. Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy.

    PubMed

    Radley-Crabb, Hannah G; Marini, Juan C; Sosa, Horacio A; Castillo, Liliana I; Grounds, Miranda D; Fiorotto, Marta L

    2014-01-01

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles. PMID:24586653

  3. Moderate beer consumption does not change early or mature atherosclerosis in mice

    PubMed Central

    Escolà-Gil, Joan Carles; Calpe-Berdiel, Laura; Ribas, Vicent; Blanco-Vaca, Francisco

    2004-01-01

    Background Although the consumption of wine in particular has been associated with a lower risk of atherothrombotic cardiovascular disease, systematic reviews differ as to the relative protective effect of beer, wine and spirits. Two previous studies showed that red wine reduces fatty streak formation (early atherosclerosis) but not mature atherosclerosis in apolipoprotein (apo) E-deficient (apoE-/-) mice. Aim of the study To determine whether a moderate beer intake would affect early and mature atherosclerotic lesion formation using control C57BL/6 and apoE-/- mice, respectively, as models. Methods Control C57BL/6 and apoE-/- mice were randomized to receive either water, ethanol, mild beer, dark beer or ethanol-free beer. The level of beer was designed to approximate the alcohol intake currently believed to be beneficial in reducing human vascular risk. Control C57BL/6 mice were fed a Western diet for 24 weeks, and apoE-/- mice a chow diet for 12 weeks. At the end of the trial period, mice were euthanized and atherosclerotic lesions quantified. Plasma lipid concentrations were also measured. Results The amount of atherosclerosis and average number of lesions in the proximal aortic region did not differ among groups in control C57BL/6 mice (p = 0.32 and p = 0.29, respectively) and apoE-/- mice (p = 0.19 and p = 0.59, respectively). No consistent differences were observed in plasma lipid and lipoprotein concentrations among water, ethanol and beer groups. Conclusions Moderate beer consumption does not change the development of early or mature atherosclerosis in mice. Our findings do not support the hypothesis of an anti-atherogenic effect of beer. Other potential protective actions of moderate beer consumption such as plaque stabilization, a reduction in plaque intrinsic thrombogenicity, or a reduction in the systemic propensity to thrombosis, remain to be studied. PMID:14725716

  4. Maturation stage enamel malformations in Amtn and Klk4 null mice.

    PubMed

    Núñez, Stephanie M; Chun, Yong-Hee P; Ganss, Bernhard; Hu, Yuanyuan; Richardson, Amelia S; Schmitz, James E; Fajardo, Roberto; Yang, Jie; Hu, Jan C-C; Simmer, James P

    2016-01-01

    Amelotin (AMTN) and kallikrein-4 (KLK4) are secreted proteins specialized for enamel biomineralization. We characterized enamel from wild-type, Amtn(-/-), Klk4(-/-), Amtn(+/-)Klk4(+/-) and Amtn(-/-)Klk4(-/-) mice to gain insights into AMTN and KLK4 functions during amelogenesis. All of the null mice were healthy and fertile. The mandibular incisors in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice were chalky-white and chipped. No abnormalities except in enamel were observed, and no significant differences were detected in enamel thickness or volume, or in rod decussation. Micro-computed tomography (μCT) maximum intensity projections localized the onset of enamel maturation in wild-type incisors distal to the first molar, but mesial to this position in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice, demonstrating a delay in enamel maturation in Amtn(-/-) incisors. Micro-CT detected significantly reduced enamel mineral density (2.5 and 2.4gHA/cm(3)) in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice respectively, compared with wild-type enamel (3.1gHA/cm(3)). Backscatter scanning electron microscopy showed that mineral density progressively diminished with enamel depth in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice. The Knoop hardness of the Amtn(-/-) outer enamel was significantly reduced relative to the wild-type and was not as hard as the middle or inner enamel. Klk4(-/-) enamel hardness was significantly reduced at all levels, but the outer enamel was significantly harder than the inner and middle enamel. Thus the hardness patterns of the Amtn(-/-) and Klk4(-/-) mice were distinctly different, while the Amtn(-/-)Klk4(-/-) outer enamel was not as hard as in the Amtn(-/-) and Klk4(-/-) mice. We conclude that AMTN and KLK4 function independently, but are both necessary for proper enamel maturation. PMID:26620968

  5. Cardiomyocyte Regeneration in the mdx Mouse Model of Nonischemic Cardiomyopathy

    PubMed Central

    Laval, Steven; Owens, William Andrew

    2015-01-01

    Endogenous regeneration has been demonstrated in the mammalian heart after ischemic injury. However, approximately one-third of cases of heart failure are secondary to nonischemic heart disease and cardiac regeneration in these cases remains relatively unexplored. We, therefore, aimed at quantifying the rate of new cardiomyocyte formation at different stages of nonischemic cardiomyopathy. Six-, 12-, 29-, and 44-week-old mdx mice received a 7 day pulse of BrdU. Quantification of isolated cardiomyocyte nuclei was undertaken using cytometric analysis to exclude nondiploid nuclei. Between 6–7 and 12–13 weeks, there was a statistically significant increase in the number of BrdU-labeled nuclei in the mdx hearts compared with wild-type controls. This difference was lost by the 29–30 week time point, and a significant decrease in cardiomyocyte generation was observed in both the control and mdx hearts by 44–45 weeks. Immunohistochemical analysis demonstrated BrdU-labeled nuclei exclusively in mononucleated cardiomyocytes. This study demonstrates cardiomyocyte regeneration in a nonischemic model of mammalian cardiomyopathy, controlling for changes in nuclear ploidy, which is lost with age, and confirms a decrease in baseline rates of cardiomyocyte regeneration with aging. While not attempting to address the cellular source of regeneration, it confirms the potential utility of innate regeneration as a therapeutic target. PMID:25749191

  6. Exopolysaccharide Produced by Lactobacillus Plantarum Induces Maturation of Dendritic Cells in BALB/c Mice

    PubMed Central

    Tang, Yanjun; Dong, Wei; Wan, Keyu; Zhang, Ligang; Li, Chun; Zhang, Lili; Liu, Ning

    2015-01-01

    Lactobacillus plantarum (L. plantarum) exopolysaccharide (EPS) is an important bioactive component in fermented functional foods. However, there is a lack of data concerning the effects of L. plantarum EPS on maturation of mouse dendritic cells (DCs). In this study, we purified L. plantarum EPS and examined its effects on cytokines production by dendritic cells in serum and intestinal fluid of BALB/c mice, then investigated its effects on phenotypic and functional maturation of mouse bone marrow-derived dendritic cells (BMDCs). Cytokines (nitric oxide, IL-12p70, IL-10 and RANTES) in serum and intestinal fluid were analyzed by enzyme linked immunosorbent assay (ELISA) after the mice received EPS for 2, 5 and 7 days, respectively. DCs derived from bone marrow of BALB/c mouse were treated with EPS, then the phenotypic maturation of BMDCs was analyzed using flow cytometer and the functional maturation of BMDCs was analyzed by ELISA, and, lastly, mixed lymphocyte proliferation was performed. We found the molecular weight of purified EPS was approximately 2.4×106 Da and it was composed of ribose, rhamnose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2:1:1:10:4:205:215. We observed that L. plantarum EPS enriched production of nitric oxide, IL-12p70 and RANTES, and decreased the secretion of IL-10 in the serum or intestinal fluid as well as in the supernatant of DCs treated with the EPS. The EPS also up-regulated the expression of MHC II and CD86 on DCs surface and promoted T cells to proliferate in vitro. Our data provide direct evidence to suggest that L. plantarum EPS can effectively induce maturation of DCs in mice. PMID:26599612

  7. Behavioral responses to and brain distribution of morphine in mature adult and aged mice

    SciTech Connect

    Burton, C.K.; Ho, I.K.; Hoskins, B.

    1986-03-01

    Mature adult (3-6 mo old) and aged (2 yr old) male ICR mice were injected with 10 to 100 mg/kg morphine, s.c. The ED50 values for running behavior (as measured using Stoelting activity monitors and having each mouse serve as its own control) representing 5 times control activity was approximately 7.5 mg/kg for aged mice and approximately 17.5 mg/kg for the mature adults. The ED50 values for analgesia 1 hr after morphine administration using the tail-flick method (max. response time = 8 sec) were approx. 70 mg/kg for the aged mice and 15 mg/kg for the mature adults. One hour after injecting /sup 3/H-morphine at doses of 30 and 100 mg/kg, 0.13 and 0.14% of the doses appeared in brains of aged and mature adult mice, respectively. Regional distribution of the morphine was the same for both age groups. Expressed as percent of total brain morphine, it was as follows: cortex, 30%; midbrain, 18%; cerebellum, 17%; medulla, 12%; pons, 9%; striatum, 8% and periaqueductal gray, 6%. Expressed as g morphine/g tissue for the 2 doses, the distribution was; periaqueductal gray, 30 and 80; striatum, 9 and 34; medulla, 6 and 20 pons; 5 and 19; cerebellum, 4 and 13; midbrain 2.5 and 8.5 and cortex, 2 and 8. These results suggest that the differences in response to morphine by the two age groups were due to age-related differences in opioid receptor populations and/or affinities.

  8. Effect of Holothuria leucospilota extracted saponin on maturation of mice oocyte and granulosa cells

    PubMed Central

    Moghadam, Fereshteh Delghandi; Baharara, Javad; Balanezhad, Saeedeh Zafar; Jalali, Mohsen; Amini, Elaheh

    2016-01-01

    Sea cucumbers saponins are triterpenoid glycosides which exert beneficial biomedical effects. This study was performed to assess the effect of saponin extracted from sea cucumber Holothuria leucospilota (H. leucospilota) on maturation of mice oocytes and granulosa cells. The germinal vesicles oocytes were collected from 6–8 weeks old Naval Medical Research Institute (NMRI) mice ovaries, randomly divided into untreated and four experimental groups and cultured In vitro. Maturation medium was supplemented with 0, 1, 2, 4 and 8 μg/ml saponin for 12 days. The rates of maturation were recorded through morphological observation by measurement of follicle diameter during treatment. After 4 days, the effects of saponin on granulosa cells were investigated by reactive oxygen species (ROS) measurement, super oxide dismutase (SOD) activity, caspase assay and tumor necrosis factor-alpha (TNF-α) expression. The oocyte maturation rate was significantly higher in treated groups (1 μg/ml). The ROS and SOD assays demonstrated the antioxidant potential of saponin. The caspase assay exhibited that optimum concentrations of saponin (1, 2 μg/ml) reduced caspase activity in granulosa cells. Flow cytometry showed that optimum concentration of saponin promoted oocyte maturation via down regulation of TNF-α as follicular degenerative factor in nursing cells. These results proposed that maturation rate were obtained after the incorporation of 1 μg/ml sea cucumber saponin. Moreover, the extracted saponin at concentrations of 1, 2 μg/ml enhanced follicle growth which is accompanied by attenuating ROS formation, elevating SOD activity and reducing TNF-α expression in granulosa cells. But, further examinations are required to understand precise mechanisms of saponin action on oocyte and granulosa cells. PMID:27168752

  9. Effect of Holothuria leucospilota extracted saponin on maturation of mice oocyte and granulosa cells.

    PubMed

    Moghadam, Fereshteh Delghandi; Baharara, Javad; Balanezhad, Saeedeh Zafar; Jalali, Mohsen; Amini, Elaheh

    2016-01-01

    Sea cucumbers saponins are triterpenoid glycosides which exert beneficial biomedical effects. This study was performed to assess the effect of saponin extracted from sea cucumber Holothuria leucospilota (H. leucospilota) on maturation of mice oocytes and granulosa cells. The germinal vesicles oocytes were collected from 6-8 weeks old Naval Medical Research Institute (NMRI) mice ovaries, randomly divided into untreated and four experimental groups and cultured In vitro. Maturation medium was supplemented with 0, 1, 2, 4 and 8 μg/ml saponin for 12 days. The rates of maturation were recorded through morphological observation by measurement of follicle diameter during treatment. After 4 days, the effects of saponin on granulosa cells were investigated by reactive oxygen species (ROS) measurement, super oxide dismutase (SOD) activity, caspase assay and tumor necrosis factor-alpha (TNF-α) expression. The oocyte maturation rate was significantly higher in treated groups (1 μg/ml). The ROS and SOD assays demonstrated the antioxidant potential of saponin. The caspase assay exhibited that optimum concentrations of saponin (1, 2 μg/ml) reduced caspase activity in granulosa cells. Flow cytometry showed that optimum concentration of saponin promoted oocyte maturation via down regulation of TNF-α as follicular degenerative factor in nursing cells. These results proposed that maturation rate were obtained after the incorporation of 1 μg/ml sea cucumber saponin. Moreover, the extracted saponin at concentrations of 1, 2 μg/ml enhanced follicle growth which is accompanied by attenuating ROS formation, elevating SOD activity and reducing TNF-α expression in granulosa cells. But, further examinations are required to understand precise mechanisms of saponin action on oocyte and granulosa cells. PMID:27168752

  10. Proteomics of Secretory-Stage and Maturation-Stage Enamel of Genetically Distinct Mice.

    PubMed

    Charone, Senda; De Lima Leite, Aline; Peres-Buzalaf, Camila; Silva Fernandes, Mileni; Ferreira de Almeida, Lucas; Zardin Graeff, Marcia Sirlene; Cardoso de Oliveira, Rodrigo; Campanelli, Ana Paula; Groisman, Sonia; Whitford, Gary Milton; Everett, Eric T; Buzalaf, Marília Afonso Rabelo

    2016-01-01

    The mechanisms by which excessive ingestion of fluoride (F) during amelogenesis leads to dental fluorosis (DF) are still not precisely known. Inbred strains of mice vary in their susceptibility to develop DF, and therefore permit the investigation of underlying molecular events influencing DF severity. We employed a proteomic approach to characterize and evaluate changes in protein expression from secretory-stage and maturation-stage enamel in 2 strains of mice with different susceptibilities to DF (A/J, i.e. 'susceptible' and 129P3/J, i.e. 'resistant'). Weanling male and female susceptible and resistant mice fed a low-F diet were divided into 2 F-water treatment groups. They received water containing 0 (control) or 50 mg F/l for 6 weeks. Plasma and incisor enamel was analyzed for F content. For proteomic analysis, the enamel proteins extracted for each group were separated by 2-dimensional electrophoresis and subsequently characterized by liquid-chromatography electrospray-ionization quadrupole time-of-flight mass spectrometry. F data were analyzed by 2-way ANOVA and Bonferroni's test (p < 0.05). Resistant mice had significantly higher plasma and enamel F concentrations when compared with susceptible mice in the F-treated groups. The proteomic results for mice treated with 0 mg F/l revealed that during the secretory stage, resistant mice had a higher abundance of proteins than their susceptible counterparts, but this was reversed during the maturation stage. Treatment with F greatly increased the number of protein spots detected in both stages. Many proteins not previously described in enamel (e.g. type 1 collagen) as well as some uncharacterized proteins were identified. Our findings reveal new insights regarding amelogenesis and how genetic background and F affect this process. PMID:26820156

  11. Linking cytoarchitecture to metabolism: sarcolemma-associated plectin affects glucose uptake by destabilizing microtubule networks in mdx myofibers

    PubMed Central

    2013-01-01

    Background Duchenne muscular dystrophy (DMD) is one of the most frequent forms of muscular disorders. It is caused by the absence of dystrophin, a core component of the sarcolemma-associated junctional complex that links the cytoskeleton to the extracellular matrix. We showed previously that plectin 1f (P1f), one of the major muscle-expressed isoforms of the cytoskeletal linker protein plectin, accumulates at the sarcolemma of DMD patients as well as of mdx mice, a widely studied animal model for DMD. Based on plectin’s dual role as structural protein and scaffolding platform for signaling molecules, we speculated that the dystrophic phenotype observed after loss of dystrophin was caused, at least to some extent, by excess plectin. Thus, we hypothesized that elimination of plectin expression in mdx skeletal muscle, while probably resulting in an overall more severe phenotype, may lead to a partial phenotype rescue. In particular, we wanted to assess whether excess sarcolemmal plectin contributes to the dysregulation of sugar metabolism in mdx myofibers. Methods We generated plectin/dystrophin double deficient (dKO) mice by breeding mdx with conditional striated muscle-restricted plectin knockout (cKO) mice. The phenotype of these mice was comparatively analyzed with that of mdx, cKO, and wild-type mice, focusing on structural integrity and dysregulation of glucose metabolism. Results We show that the accumulation of plectin at the sarcolemma of mdx muscle fibers hardly compensated for their loss of structural integrity. Instead, it led to an additional metabolic deficit by impairing glucose uptake. While dKO mice suffered from an overall more severe form of muscular dystrophy compared to mdx or plectin-deficient mice, sarcolemmal integrity as well as glucose uptake of their myofibers were restored to normal levels upon ablation of plectin. Furthermore, microtubule (MT) networks in intact dKO myofibers, including subsarcolemmal areas, were found to be more robust

  12. Maternal immune activation differentially impacts mature and adult-born hippocampal neurons in male mice.

    PubMed

    Zhang, Zhi; van Praag, Henriette

    2015-03-01

    Schizophrenia is associated with deficits in the hippocampus, a brain area important for learning and memory. The dentate gyrus (DG) of the hippocampus develops both before and after birth. To study the relative contribution of mature and adult-born DG granule cells to disease etiology, we compared both cell populations in a mouse model of psychiatric illness resulting from maternal immune activation. Polyriboinosinic-polyribocytidilic acid (PolyIC, 5mg/kg) or saline was given on gestation day 15 to pregnant female C57Bl/6 mice. Male offspring (n=105), was administered systemic bromodeoxyuridine (BrdU, 50mg/kg) (n=52) or intracerebral retroviral injection into the DG (n=53), to label dividing cells at one month of age. Two months later behavioral tests were performed to evaluate disease phenotype. Immunohistochemistry and whole-cell patch clamping were used to assess morphological and physiological characteristics of DG cells. Three-month-old PolyIC exposed male offspring exhibited deficient pre-pulse inhibition, spatial maze performance and motor coordination, as well as increased depression-like behavior. Histological analysis showed reduced DG volume and parvalbumin positive interneuron number. Both mature and new hippocampal neurons showed modifications in intrinsic properties such as increased input resistance and lower current threshold, and decreased action potential number. Reduced GABAergic inhibitory transmission was observed only in mature DG neurons. Differential impairments in mature DG cells and adult-born new neurons may have implications for behavioral deficits associated with maternal immune activation. PMID:25449671

  13. Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice.

    PubMed

    Watanabe, Shigeru; Yamamori, Saori; Otsuka, Shintaro; Saito, Masanori; Suzuki, Eiji; Kataoka, Masakazu; Miyaoka, Hitoshi; Takahashi, Masami

    2015-09-01

    Snap25(S187A/S187A) mouse is a knock-in mouse with a single amino acid substitution at a protein kinase C-dependent phosphorylation site of the synaptosomal-associated protein of 25 kDa (SNAP-25), which is a target-soluble NSF attachment protein receptor (t-SNARE) protein essential for neurotransmitter release. Snap25(S187A/S187A) mice exhibit several distinct phenotypes, including reductions in dopamine and serotonin release in the brain, anxiety-like behavior, and cognitive dysfunctions. Homozygous mice show spontaneous epileptic convulsions, and about 15% of the mice die around three weeks after birth. The remaining mice survive for almost two years and exhibit spontaneous recurrent seizures throughout their lifetime. Here, we conducted long-term continuous video electroencephalogram recording of the mice and analyzed the process of epileptogenesis and epileptic maturation in detail. Spikes and slow-wave discharges (SWDs) were observed in the cerebral cortex and thalamus before epileptic convulsions began. SWDs showed several properties similar to those observed in absence seizures including (1) lack of in the hippocampus, (2) movement arrest during SWDs, and (3) inhibition by ethosuximide. Multiple generalized seizures occurred in all homozygous mice around three weeks after birth. However, seizure generation stopped within several days, and a seizure-free latent period began. Following a spike-free quiet period, the number of spikes increased gradually, and epileptic seizures reappeared. Subsequently, spontaneous seizures occurred cyclically throughout the life of the mice, and several progressive changes in seizure frequency, seizure duration, seizure cycle interval, seizure waveform, and the number and waveform of epileptic discharges during slow-wave sleep occurred with different time courses over 10 weeks. Anxiety-related behaviors appeared suddenly within three days after epileptic seizures began and were delayed markedly by oral administration of

  14. Scrapie affects the maturation cycle and immune complex trapping by follicular dendritic cells in mice.

    PubMed

    McGovern, Gillian; Mabbott, Neil; Jeffrey, Martin

    2009-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurological disorders of man and animals, characterised by abnormal disease-associated prion protein (PrP(d)) accumulations in the brain and lymphoreticular system (LRS). Prior to neuroinvasion, TSE agents often accumulate to high levels within the LRS, apparently without affecting immune function. However, our analysis of scrapie-affected sheep shows that PrP(d) accumulations within the LRS are associated with morphological changes to follicular dendritic cells (FDCs) and tingible body macrophages (TBMs). Here we examined FDCs and TBMs in the mesenteric lymph nodes (MLNs) of scrapie-affected mice by light and electron microscopy. In MLNs from uninfected mice, FDCs could be morphologically categorised into immature, mature and regressing forms. However, in scrapie-affected MLNs this maturation cycle was adversely affected. FDCs characteristically trap and retain immune complexes on their surfaces, which they display to B-lymphocytes. In scrapie-affected MLNs, some FDCs were found where areas of normal and abnormal immune complex retention occurred side by side. The latter co-localised with PrP(d) plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrP(d) accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrP(d). Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrP(d) accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function. PMID:19997557

  15. The role of cilostazol, a phosphodiesterase 3 inhibitor, on oocyte maturation and subsequent pregnancy in mice.

    PubMed

    Li, Min; Yu, Yang; Yan, Jie; Yan, Li-Ying; Zhao, Yue; Li, Rong; Liu, Ping; Hsueh, Aaron J; Qiao, Jie

    2012-01-01

    It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug. PMID:22292006

  16. Reduced Connexin26 in the Mature Cochlea Increases Susceptibility to Noise-Induced Hearing Lossin Mice.

    PubMed

    Zhou, Xing-Xing; Chen, Sen; Xie, Le; Ji, Yu-Zi; Wu, Xia; Wang, Wen-Wen; Yang, Qi; Yu, Jin-Tao; Sun, Yu; Lin, Xi; Kong, Wei-Jia

    2016-01-01

    Connexin26 (Cx26, encoded by GJB2) mutations are the most common cause of non-syndromic deafness. GJB2 is thought to be involved in noise-induced hearing loss (NIHL). However, the role of Cx26 in NIHL is still obscure. To explore the association between Cx26 and NIHL, we established a Cx26 knockdown (KD) mouse model by conditional knockdown of Cx26 at postnatal day 18 (P18), and then we observed the auditory threshold and morphologic changes in these mice with or without noise exposure. The Cx26 KD mice did not exhibit substantial hearing loss and hair cell degeneration, while the Cx26 KD mice with acoustic trauma experienced higher hearing loss than simple noise exposure siblings and nearly had no recovery. Additionally, extensive outer hair cell loss and more severe destruction of the basal organ of Corti were observed in Cx26 KD mice after noise exposure. These data indicate that reduced Cx26 expression in the mature mouse cochlea may increase susceptibility to noise-induced hearing loss and facilitate the cell degeneration in the organ of Corti. PMID:26927086

  17. Hypothyroidism Impairs Human Stem Cell-Derived Pancreatic Progenitor Cell Maturation in Mice.

    PubMed

    Bruin, Jennifer E; Saber, Nelly; O'Dwyer, Shannon; Fox, Jessica K; Mojibian, Majid; Arora, Payal; Rezania, Alireza; Kieffer, Timothy J

    2016-05-01

    Pancreatic progenitors derived from human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating diabetes and are currently being tested in clinical trials. Yet, how the milieu of pancreatic progenitor cells, including exposure to different factors after transplant, may influence their maturation remains unclear. Here, we examined the effect of thyroid dysregulation on the development of hESC-derived progenitor cells in vivo. Hypothyroidism was generated in SCID-beige mice using an iodine-deficient diet containing 0.15% propyl-2-thiouracil, and hyperthyroidism was generated by addition of L-thyroxine (T4) to drinking water. All mice received macroencapsulated hESC-derived progenitor cells, and thyroid dysfunction was maintained for the duration of the study ("chronic") or for 4 weeks posttransplant ("acute"). Acute hyperthyroidism did not affect graft function, but acute hypothyroidism transiently impaired human C-peptide secretion at 16 weeks posttransplant. Chronic hypothyroidism resulted in severely blunted basal human C-peptide secretion, impaired glucose-stimulated insulin secretion, and elevated plasma glucagon levels. Grafts from chronic hypothyroid mice contained fewer β-cells, heterogenous MAFA expression, and increased glucagon(+) and ghrelin(+) cells compared to grafts from euthyroid mice. Taken together, these data suggest that long-term thyroid hormone deficiency may drive the differentiation of human pancreatic progenitor cells toward α- and ε-cell lineages at the expense of β-cell formation. PMID:26740603

  18. Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF.

    PubMed

    Swee, Lee Kim; Tardivel, Aubry; Schneider, Pascal; Rolink, Antonius

    2010-06-15

    BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment. PMID:20350570

  19. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy

    PubMed Central

    Grounds, Miranda D.; Radley, Hannah G.; Lynch, Gordon S.; Nagaraju, Kanneboyina; De Luca, Annamaria

    2008-01-01

    This review discusses various issues to consider when developing standard operating procedures for pre-clinical studies in the mdx mouse model of Duchenne muscular dystrophy (DMD). The review describes and evaluates a wide range of techniques used to measure parameters of muscle pathology in mdx mice and identifies some basic techniques that might comprise standardised approaches for evaluation. While the central aim is to provide a basis for the development of standardised procedures to evaluate efficacy of a drug or a therapeutic strategy, a further aim is to gain insight into pathophysiological mechanisms in order to identify other therapeutic targets. The desired outcome is to enable easier and more rigorous comparison of pre-clinical data from different laboratories around the world, in order to accelerate identification of the best pre-clinical therapies in the mdx mouse that will fast-track translation into effective clinical treatments for DMD. PMID:18499465

  20. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

    PubMed

    Cao, Yun; Bender, Ingrid K; Konstantinidis, Athanasios K; Shin, Soon Cheon; Jewell, Christine M; Cidlowski, John A; Schleimer, Robert P; Lu, Nick Z

    2013-02-28

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  1. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans

    PubMed Central

    Cao, Yun; Bender, Ingrid K.; Konstantinidis, Athanasios K.; Shin, Soon Cheon; Jewell, Christine M.; Cidlowski, John A.; Schleimer, Robert P.

    2013-01-01

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  2. Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx, Largemyd and mdx/Largemyd

    PubMed Central

    Martins-Bach, Aurea B.; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C. M.; Almeida, Camila F.; Caldeira, Waldir; Ribeiro, Alberto F.; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G.; Vainzof, Mariz

    2015-01-01

    Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research

  3. Quantitative T2 combined with texture analysis of nuclear magnetic resonance images identify different degrees of muscle involvement in three mouse models of muscle dystrophy: mdx, Largemyd and mdx/Largemyd.

    PubMed

    Martins-Bach, Aurea B; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C M; Almeida, Camila F; Caldeira, Waldir; Ribeiro, Alberto F; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G; Vainzof, Mariz

    2015-01-01

    Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant-T2-measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research. PMID

  4. Introduction of a human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy

    PubMed Central

    Chandrasekharan, Kumaran; Yoon, Jung Hae; Xu, Ying; deVries, Sarah; Camboni, Marybeth; Janssen, Paulus M.L.; Varki, Ajit; Martin, Paul T.

    2010-01-01

    The evolution of humans included introduction of an inactivating deletion in the CMAH gene, which eliminated biosynthesis of N-glycolylneuraminic acid from all human cells. Here we show that this human-specific sialylation change contributes to the marked discrepancy in phenotype between the mdx mouse model for Duchenne muscular dystrophy (DMD) and the human disease. Despite lacking dystrophin protein in almost all muscle cells, mdx mice show slower development, relative to overall lifespan, or reduced severity of a number of clinically relevant disease phenotypes compared to DMD patients. This is especially true for loss of ambulation, cardiac and respiratory muscle weakness, and loss of lifespan, all major phenotypes contributing to DMD morbidity and mortality. All these phenotypes occur at an earlier age or to a greater degree in mdx mice bearing a human-like mutation in the mouse Cmah gene. Altered phenotypes correlate with changes in two mechanisms; reduced strength and expression of the dystrophin-associated glycoprotein complex and increased activation of complement. Activation of complement may be driven by the increased expression of anti-Neu5Gc antibodies in Cmah−/−mdx animals and ultimately by uptake of N-glycolylneuraminic acid, a foreign glycan in humans and Cmah-deficient mice, from dietary sources. Cmah-deficient mdx mice represent a new small animal model for DMD that better approximates the human glycome and its contributions to muscular dystrophy. PMID:20668298

  5. Synaptic underpinnings of altered hippocampal function in glutaminase-deficient mice during maturation.

    PubMed

    Gaisler-Salomon, Inna; Wang, Yvonne; Chuhma, Nao; Zhang, Hong; Golumbic, Yaela N; Mihali, Andra; Arancio, Ottavio; Sibille, Etienne; Rayport, Stephen

    2012-05-01

    Glutaminase-deficient mice (GLS1 hets), with reduced glutamate recycling, have a focal reduction in hippocampal activity, mainly in CA1, and manifest behavioral and neurochemical phenotypes suggestive of schizophrenia resilience. To address the basis for the hippocampal hypoactivity, we examined synaptic plastic mechanisms and glutamate receptor expression. Although baseline synaptic strength was unaffected in Schaffer collateral inputs to CA1, we found that long-term potentiation was attenuated. In wild-type (WT) mice, GLS1 gene expression was highest in the hippocampus and cortex, where it was reduced by about 50% in GLS1 hets. In other brain regions with lower WT GLS1 gene expression, there were no genotypic reductions. In adult GLS1 hets, NMDA receptor NR1 subunit gene expression was reduced, but not AMPA receptor GluR1 subunit gene expression. In contrast, juvenile GLS1 hets showed no reductions in NR1 gene expression. In concert with this, adult GLS1 hets showed a deficit in hippocampal-dependent contextual fear conditioning, whereas juvenile GLS1 hets did not. These alterations in glutamatergic synaptic function may partly explain the hippocampal hypoactivity seen in the GLS1 hets. The maturity-onset reduction in NR1 gene expression and in contextual learning supports the premise that glutaminase inhibition in adulthood should prove therapeutic in schizophrenia. PMID:22431402

  6. Spinal cord maturation and locomotion in mice with an isolated cortex.

    PubMed

    Han, Q; Feng, J; Qu, Y; Ding, Y; Wang, M; So, K-F; Wu, W; Zhou, L

    2013-12-01

    The spinal cord plays a key role in motor behavior. It relays major sensory information, receives afferents from supraspinal centers and integrates movement in the central pattern generators. Spinal motor output is controlled via corticofugal pathways including corticospinal and cortico-subcortical projections. Spinal cord injury damages descending supraspinal as well as ascending sensory pathways. In adult rodent models, plasticity of the spinal cord is thought to contribute to functional recovery. How much spinal cord function depends on cortical input is not well known. Here, we address this question using Celsr3/Foxg1 mice, in which cortico-subcortical connections (including corticospinal tract (CST) and the terminal sensory pathway, the thalamocortical tract) are genetically ablated during early development. Although Celsr3/Foxg1 mice are able to eat, walk, climb on grids and swim, open-field tests showed them to be hyperactive. When compared with normal littermates, mutant animals had reduced number of spinal motor neurons, with atrophic dendritic trees. Furthermore, motor axon terminals were decreased in number, and this was confirmed by electromyography. The number of cholinergic, calbindin, and calretinin-positive interneurons was moderately increased in the mutant spinal cord, whereas that of reelin and parvalbumin-positive interneurons was unchanged. As far as we know, our study provides the first genetic evidence that the spinal motor network does not mature fully in the absence of corticofugal connections, and that some motor function is preserved despite congenital absence of the CST. PMID:24012835

  7. Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice.

    PubMed

    Kueakhai, Pornanan; Changklungmoa, Narin; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Itagaki, Tadashi; Sobhon, Prasert

    2015-10-01

    In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immuno-blotting to react with the native FgCatL1 in the extract of newly excysted juveniles (NEJ), 4-week-old juveniles and the ES products of 4 week-old juveniles. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune response, respectively, it was found that both Th1 and Th2 responses were significantly increased in rproFgCatL1- and rmatFgCatL1-immunized groups compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rmatFgCatL1-immunized group showed a significant decrease when compared to rproFgCatL1-immunized group, indicating that rmatFgCatL1-vaccinated mice had reduced liver parenchyma damage. The pathological lesions of liver in vaccinated groups were significantly decreased when compared with control groups. This study indicates that r

  8. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice

    PubMed Central

    Fu, Chun; Begum, Khurshida; Jordan, Philip W.; He, Yan; Overbeek, Paul A.

    2016-01-01

    After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line. PMID:27486799

  9. A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.

    PubMed

    Arpke, Robert W; Darabi, Radbod; Mader, Tara L; Zhang, Yu; Toyama, Akira; Lonetree, Cara-Lin; Nash, Nardina; Lowe, Dawn A; Perlingeiro, Rita C R; Kyba, Michael

    2013-08-01

    Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice, we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx(4Cv) mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx(4Cv) mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx(4Cv) recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function and the utility of the NSG-mdx(4Cv) model for studies on muscle regeneration and Duchenne muscular dystrophy therapy. PMID:23606600

  10. Long-term treatment with naproxcinod significantly improves skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy.

    PubMed

    Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Van Der Meulen, Jack H; Yu, Qing; Phadke, Aditi; Miller, Brittany K; Gordish-Dressman, Heather; Ongini, Ennio; Miglietta, Daniela; Nagaraju, Kanneboyina

    2014-06-15

    In Duchenne muscular dystrophy (DMD) patients and the mouse model of DMD, mdx, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to functional impairments. Previous studies have shown that nitric oxide (NO) donation associated with anti-inflammatory action has beneficial effects in dystrophic mouse models. In this study, we have systematically investigated the effects of naproxcinod, an NO-donating naproxen derivative, on the skeletal and cardiac disease phenotype in mdx mice. Four-week-old mdx and C57BL/10 mice were treated with four different concentrations (0, 10, 21 and 41 mg/kg) of naproxcinod and 0.9 mg/kg of prednisolone in their food for 9 months. All mice were subjected to twice-weekly treadmill sessions, and functional and behavioral parameters were measured at 3, 6 and 9 months of treatment. In addition, we evaluated in vitro force contraction, optical imaging of inflammation, echocardiography and blood pressure (BP) at the 9-month endpoint prior to sacrifice. We found that naproxcinod treatment at 21 mg/kg resulted in significant improvement in hindlimb grip strength and a 30% decrease in inflammation in the fore- and hindlimbs of mdx mice. Furthermore, we found significant improvement in heart function, as evidenced by improved fraction shortening, ejection fraction and systolic BP. In addition, the long-term detrimental effects of prednisolone typically seen in mdx skeletal and heart function were not observed at the effective dose of naproxcinod. In conclusion, our results indicate that naproxcinod has significant potential as a safe therapeutic option for the treatment of muscular dystrophies. PMID:24463621

  11. Long-term treatment with naproxcinod significantly improves skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy

    PubMed Central

    Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Van Der Meulen, Jack H; Yu, Qing; Phadke, Aditi; Miller, Brittany K; Gordish-Dressman, Heather; Ongini, Ennio; Miglietta, Daniela; Nagaraju, Kanneboyina

    2014-01-01

    In Duchenne muscular dystrophy (DMD) patients and the mouse model of DMD, mdx, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to functional impairments. Previous studies have shown that nitric oxide (NO) donation associated with anti-inflammatory action has beneficial effects in dystrophic mouse models. In this study, we have systematically investigated the effects of naproxcinod, an NO-donating naproxen derivative, on the skeletal and cardiac disease phenotype in mdx mice. Four-week-old mdx and C57BL/10 mice were treated with four different concentrations (0, 10, 21 and 41 mg/kg) of naproxcinod and 0.9 mg/kg of prednisolone in their food for 9 months. All mice were subjected to twice-weekly treadmill sessions, and functional and behavioral parameters were measured at 3, 6 and 9 months of treatment. In addition, we evaluated in vitro force contraction, optical imaging of inflammation, echocardiography and blood pressure (BP) at the 9-month endpoint prior to sacrifice. We found that naproxcinod treatment at 21 mg/kg resulted in significant improvement in hindlimb grip strength and a 30% decrease in inflammation in the fore- and hindlimbs of mdx mice. Furthermore, we found significant improvement in heart function, as evidenced by improved fraction shortening, ejection fraction and systolic BP. In addition, the long-term detrimental effects of prednisolone typically seen in mdx skeletal and heart function were not observed at the effective dose of naproxcinod. In conclusion, our results indicate that naproxcinod has significant potential as a safe therapeutic option for the treatment of muscular dystrophies. PMID:24463621

  12. Variable maturation and oviposition by female Schistosoma japonicum in mice: the effects of irradiation of the host prior to infection

    SciTech Connect

    Cheever, A.W.; Duvall, R.H.

    1987-11-01

    The maturation of female Schistosoma japonicum was found to vary greatly within each of two Philippine strains of this parasite and some females did not contain uterine eggs 7 to 15 weeks after infection while others contained numerous eggs before the fifth week of infection. It was found that female worms containing less than 20 uterine eggs contributed little to the accumulation of eggs in the tissues of infected mice. Such worms also generally appeared to be immature. The variable rate of maturation of worms is likely to have profound effects on the immune reactions of mice as well as on the pathologic response to infection. Systematic delay in oviposition was serendipitously found in worms from mice which had been irradiated for other purposes prior to exposure to S. japonicum, and from the fourth to the sixth week after infection egg production by worms in irradiated mice lagged well behind that in intact mice. Seven to 10 weeks after infection these worms were laying normal numbers of eggs, as judged by egg passage per worm pair in the feces and the accumulation of eggs in the tissues. S. mansoni developed normally in irradiated mice.

  13. Bile acid-FXRα pathways regulate male sexual maturation in mice.

    PubMed

    Baptissart, Marine; Martinot, Emmanuelle; Vega, Aurélie; Sédes, Lauriane; Rouaisnel, Betty; de Haze, Angélique; Baron, Silvère; Schoonjans, Kristina; Caira, Françoise; Volle, David H

    2016-04-12

    The bile acid receptor Farnesol-X-Receptor alpha (FRXα) is a member of the nuclear receptor superfamily. FRXα is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. In adult, short term treatment (12 hours) with FRXα agonist inhibits the expression of steroidogenic genes via the induction of the Small heterodimer partner (SHP). However the consequences of FRXα activation on testicular pathophysiology have never been evaluated. We demonstrate here that mice fed a diet supplemented with bile acid during pubertal age show increased incidence of infertility. This is associated with altered differentiation and increase apoptosis of germ cells due to lower testosterone levels. At the molecular level, next to the repression of basal steroidogenesis via the induction expression of Shp and Dax-1, two repressors of steroidogenesis, the main action of the BA-FRXα signaling is through lowering the Leydig cell sensitivity to the hypothalamo-pituitary axis, the main regulator of testicular endocrine function. In conclusion, BA-FRXα signaling is a critical actor during sexual maturation. PMID:26848619

  14. The small heterodimer partner is a gonadal gatekeeper of sexual maturation in male mice

    PubMed Central

    Volle, David H.; Duggavathi, Rajesha; Magnier, Benjamin C.; Houten, Sander M.; Cummins, Carolyn L.; Lobaccaro, Jean-Marc A.; Verhoeven, Guido; Schoonjans, Kristina; Auwerx, Johan

    2007-01-01

    The small heterodimer partner (SHP) is an atypical nuclear receptor known mainly for its role in bile acid homeostasis in the enterohepatic tract. We explore here the role of SHP in the testis. SHP is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. SHP there inhibits the expression of steroidogenic genes, on the one hand by inhibiting the expression of the nuclear receptors steroidogenic factor-1 and liver receptor homolog-1 (lrh-1), and on the other hand by directly repressing the transcriptional activity of LRH-1. Consequently, in SHP knockout mice, testicular testosterone synthesis is increased independently of the hypothalamus–pituitary axis. Independent of its action on androgen synthesis, SHP also determines the timing of germ cell differentiation by controlling testicular retinoic acid metabolism. Through the inhibition of the transcriptional activity of retinoic acid receptors, SHP controls the expression of stimulated by retinoic acid gene 8 (stra8)—a gene that is indispensable for germ cell meiosis and differentiation. Together, our data demonstrate new roles for SHP in testicular androgen and retinoic acid metabolism, making SHP a testicular gatekeeper of the timing of male sexual maturation. PMID:17289919

  15. Bile acid-FXRα pathways regulate male sexual maturation in mice

    PubMed Central

    Vega, Aurélie; Sédes, Lauriane; Rouaisnel, Betty; de Haze, Angélique; Baron, Silvère; Schoonjans, Kristina; Caira, Françoise; Volle, David H.

    2016-01-01

    The bile acid receptor Farnesol-X-Receptor alpha (FRXα) is a member of the nuclear receptor superfamily. FRXα is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. In adult, short term treatment (12 hours) with FRXα agonist inhibits the expression of steroidogenic genes via the induction of the Small heterodimer partner (SHP). However the consequences of FRXα activation on testicular pathophysiology have never been evaluated. We demonstrate here that mice fed a diet supplemented with bile acid during pubertal age show increased incidence of infertility. This is associated with altered differentiation and increase apoptosis of germ cells due to lower testosterone levels. At the molecular level, next to the repression of basal steroidogenesis via the induction expression of Shp and Dax-1, two repressors of steroidogenesis, the main action of the BA-FRXα signaling is through lowering the Leydig cell sensitivity to the hypothalamo-pituitary axis, the main regulator of testicular endocrine function. In conclusion, BA-FRXα signaling is a critical actor during sexual maturation. PMID:26848619

  16. Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients

    PubMed Central

    Hathout, Yetrib; Marathi, Ramya L.; Rayavarapu, Sree; Zhang, Aiping; Brown, Kristy J.; Seol, Haeri; Gordish-Dressman, Heather; Cirak, Sebahattin; Bello, Luca; Nagaraju, Kanneboyina; Partridge, Terry; Hoffman, Eric P.; Takeda, Shin'ichi; Mah, Jean K.; Henricson, Erik; McDonald, Craig

    2014-01-01

    It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials. PMID:25027324

  17. Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients.

    PubMed

    Hathout, Yetrib; Marathi, Ramya L; Rayavarapu, Sree; Zhang, Aiping; Brown, Kristy J; Seol, Haeri; Gordish-Dressman, Heather; Cirak, Sebahattin; Bello, Luca; Nagaraju, Kanneboyina; Partridge, Terry; Hoffman, Eric P; Takeda, Shin'ichi; Mah, Jean K; Henricson, Erik; McDonald, Craig

    2014-12-15

    It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials. PMID:25027324

  18. Structural and functional maturation of distal femoral cartilage and bone during postnatal development and growth in humans and mice.

    PubMed

    Chan, Elaine F; Harjanto, Ricky; Asahara, Hiroshi; Inoue, Nozomu; Masuda, Koichi; Bugbee, William D; Firestein, Gary S; Hosalkar, Harish S; Lotz, Martin K; Sah, Robert L

    2012-04-01

    The size and shape of joints markedly affect their biomechanical properties, but the macroscopic 3-dimensional (3-D) mechanism and extent of cartilage and joint maturation during normal growth are largely unknown. This study qualitatively illustrates the development of the bone-cartilage interface in the knee during postnatal growth in humans and C57BL/6 wild-type mice, quantitatively defines the 3-D shape using statistical shape modeling, and assesses growth strain rates in the mouse distal femur. Accurate quantification of the cartilage-bone interface geometry is imperative for furthering the understanding of the macroscopic mechanisms of cartilage maturation and overall joint development. PMID:22480467

  19. Granulocyte-Colony Stimulating Factor Improves MDX Mouse Response to Peripheral Nerve Injury

    PubMed Central

    Simões, Gustavo Ferreira; de Oliveira, Alexandre Leite Rodrigues

    2012-01-01

    Background G-CSF has been shown to increase neuronal survival, which may positively influence the spinal cord microenvironment during the course of muscular dystrophies. Methodology/Principal Findings Male MDX mice that were six weeks of age received a left sciatic nerve transection and were treated with intraperitoneal injections of 200 µg/kg/day of G-CSF 7 days before and 7 days after the transection. The axotomy was performed after the cycles of muscular degeneration/regeneration, consistent with previous descriptions of this model of muscular dystrophy. C57BL/10 mice were used as control subjects. Seven days after the surgery, the animals were sacrificed and their lumbar spinal cords were processed for immunohistochemistry (anti-MHC I, anti-Synaptophysin, anti-GFAP and anti-IBA-1) and transmission electron microscopy. MHC I expression increased in both strains of mice after the axotomy. Nevertheless, the MDX mice displayed a significantly smaller MHC I upregulation than the control mice. Regarding GFAP expression, the MDX mice showed a stronger astrogliosis compared with the C57BL/10 mice across all groups. Both groups that were treated with G-CSF demonstrated preservation of synaptophysin expression compared with the untreated and placebo groups. The quantitative analysis of the ultrastructural level showed a preservation of the synaptic covering for the both groups that were treated with G-CSF and the axotomized groups showed a smaller loss of synaptic contact in relation to the treated groups after the lesion. Conclusions/Significance The reduction of active inputs to the alpha-motoneurons and increased astrogliosis in the axotomized and control groups may be associated with the cycles of muscle degeneration/regeneration that occur postnatally. The G-CSF treated group showed a preservation of the spinal cord microenvironment after the lesion. Moreover, the increase of MHC I expression in the MDX mice that were treated with G-CSF may indicate that this drug

  20. Effect of insulin supplementation on in vitro maturation of pre-antral follicles from adult and pre-pubertal mice.

    PubMed

    Nath, Amar; Hakim, Bilal Ahmad; Rajender, Singh; Singh, Kavita; Sachdev, Monika; Konwar, Rituraj

    2016-05-01

    This study was aimed to determine the impact of insulin concentrations on in vitro pre-antral follicle growth, survival, antrum formation rate, and retrieval of mature oocytes in mice. Mice pre-antral follicle growth were recorded on days 2, 4, 6, 8, 10, and 12 in α-modified essential media (α-MEM) supplemented with insulin concentrations of 6, 8, and 10 μg/ml along with 10% FBS, 100 mIU/ml follicle stimulating hormone, 10 mIU/ml luteinizing hormone, 100 μg/ml penicillin, and 50 μg/ml streptomycin. After 12 d of growth in vitro, follicles were allowed to mature for 16-18 h in α-MEM supplemented with 1.5 IU/ml human chorionic gonadotrophin (hCG) and 5 ng/ml epidermal growth factor (EGF). The initial diameter (54.86 ± 2.5 μm) of mice oocyte progressively increased in all the three insulin concentration groups and attained a maximum size on day 12 (71.90 ± 2.8 μm). Supplementation with higher concentrations of insulin (both 8 and 10 μg/ml) significantly enhanced antrum formation without effecting the oocyte diameter and percent retrieval of mature oocyte in all the three concentration groups. Both in vitro cultured as well as in vivo collected follicles and oocytes showed similar localization and expression of oocyte maturation markers SAS1B and GDF9. Insulin concentration of 8 μg/ml was found to be optimal for in vitro follicle culture of adult mice (42-49 d). Optimized follicle culture conditions were also assessed successfully with pre-pubertal mice (12-14 d); however, adult mice showed higher follicle survival, antrum formation, and more mature oocytes production in comparison to pre-pubertal mice. PMID:26956357

  1. Impairment of Oligodendroglia Maturation Leads to Aberrantly Increased Cortical Glutamate and Anxiety-Like Behaviors in Juvenile Mice

    PubMed Central

    Chen, Xianjun; Zhang, Weiguo; Li, Tao; Guo, Yu; Tian, Yanping; Wang, Fei; Liu, Shubao; Shen, Hai-Ying; Feng, Yue; Xiao, Lan

    2015-01-01

    Adolescence is the critical time for developing proper oligodendrocyte (OL)-neuron interaction and the peak of onset for many cognitive diseases, among which anxiety disorders display the highest prevalence. However, whether impairment of de novo OL development causes neuronal abnormalities and contributes to the early onset of anxiety phenotype in childhood still remains unexplored. In this study, we tested the hypothesis that defects in OL maturation manifests cortical neuron function and leads to anxiety-like behaviors in juvenile mice. We report here that conditional knockout of the Olig2 gene (Olig2 cKO) specifically in differentiating OLs in the mouse brain preferentially impaired OL maturation in the gray matter of cerebral cortex. Interestingly, localized proton magnetic resonance spectroscopy revealed that Olig2 cKO mice displayed abnormally elevated cortical glutamate levels. In addition, transmission electron microscopy demonstrated increased vesicle density in excitatory glutamatergic synapses in the cortex of the Olig2 cKO mice. Moreover, juvenile Olig2 cKO mice exhibited anxiety-like behaviors and impairment in behavioral inhibition. Taken together, our results suggest that impaired OL development affects glutamatergic neuron function in the cortex and causes anxiety-related behaviors in juvenile mice. These discoveries raise an intriguing possibility that OL defects may be a contributing mechanism for the onset of anxiety in childhood. PMID:26696827

  2. Effect of prenatal glucocorticoid on fetal lung ultrastructural maturation in hyt/hyt mice with primary hypothyroidism.

    PubMed

    Ansari, M A; de Mello, D E; Devaskar, U P

    2000-01-01

    Glucocorticoids (GC) and thyroid hormones (TH) accelerate fetal lung maturation. Though GC are used clinically, the mechanisms of GC-induced fetal lung maturity remain unclear. Prenatal GC increase fetal TH activity in humans and in animals. Thus, it is possible that increased fetal TH activity after prenatal GC plays a role in accelerating fetal lung maturation. However, this hypothesis has remained untested due to the lack of a suitable animal model. In the hyt/hyt mouse primary hypothyroidism occurs due to a point mutation in the beta subunit of the thyroid-stimulating hormone receptor of the thyroid gland, and it is transmitted in an autosomal recessive manner. We studied the effect of maternal betamethasone on fetal lung ultrastructure in hyt/hyt (hypothyroid) and Balb-c (euthyroid) mice. Hypothyroid mice were made euthyroid by T3 supplementation and mated to carry hypothyroid pups. Vehicle (n = 6) or betamethasone (n = 6) was injected intraperitoneally twice daily into the doe on days 16 and 17 of gestation. Fetal lungs on 18 days of gestation were subjected to ultrastructural morphometric analysis. The number of lamellar bodies per type II cell increased after betamethasone in Balb-c (2.10+/-0.31 vs. 3.43+/-0.37) and hyt/hyt (0.77+/-0.28 vs. 3.85+/-0.26) mice. The alveolar-to-parenchymal ratio was less in the vehicle-treated hyt/hyt (0.082+/-0.024) as compared with the vehicle-treated Balb-c (0.30+/-0.05) mice, while prenatal betamethasone increased the alveolar-to-parenchymal ratio in the hyt/hyt (0.227+/-0.034) but not in the Balb-c (0.26+/-0.04) mice. The glycogen-to-nucleus ratio was higher in betamethasone-treated hyt/hyt mice (1.46+/-0.20) when compared to vehicle-treated hyt/hyt (0.89+/-0.14) or Balb-c (1.01+/-0.17) or betamethasone-treated Balb-c (0.81+/-0.13) mice. Though tubular myelin was readily apparent in the airspace lumen of betamethasone-treated Balb-c mice, it was absent in betamethasone-treated hyt/hyt fetal lungs. We conclude that fetal

  3. Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.

    PubMed

    Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Aydiner, Fulya; Sasson, Isaac; Ilbay, Orkan; Sakkas, Denny; Lowther, Katie M; Mehlmann, Lisa M; Seli, Emre

    2012-08-15

    Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab-knockout mice. Although Epab(-/-) males and Epab(+/-) of both sexes were fertile, Epab(-/-) female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab(-/-) oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Ccnb1 (cyclin B1) and Dazl (deleted in azoospermia-like) mRNAs. Microinjection of Epab mRNA into Epab(-/-) germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab(-/-) mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding the EGF (epidermal growth factor)-like growth factors Areg (amphiregulin), Ereg (epiregulin) and Btc (betacellulin), and their downstream regulators, Ptgs2 (prostaglandin synthase 2), Has2 (hyaluronan synthase 2) and Tnfaip6 (tumour necrosis factor α-induced protein 6). The findings from the present study indicate that EPAB is necessary for oogenesis, folliculogenesis and female fertility in mice. PMID:22621333

  4. Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice

    PubMed Central

    Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Aydiner, Fulya; Sasson, Isaac; Ilbay, Orkan; Sakkas, Denny; Lowther, Katie M.; Mehlmann, Lisa M.; Seli, Emre

    2014-01-01

    Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab-knockout mice. Although Epab−/− males and Epab+/− of both sexes were fertile, Epab−/− female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab−/− oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Ccnb1 (cyclin B1) and Dazl (deleted in azoospermia-like) mRNAs. Microinjection of Epab mRNA into Epab−/− germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab−/− mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding the EGF (epidermal growth factor)-like growth factors Areg (amphiregulin), Ereg (epiregulin) and Btc (betacellulin), and their downstream regulators, Ptgs2 (prostaglandin synthase 2), Has2 (hyaluronan synthase 2) and Tnfaip6 (tumour necrosis factor α-induced protein 6). The findings from the present study indicate that EPAB is necessary for oogenesis, folliculogenesis and female fertility in mice. PMID:22621333

  5. A Mathematical Model of Skeletal Muscle Disease and Immune Response in the mdx Mouse

    PubMed Central

    Evans, Nicholas P.; Grange, Robert W.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disease that results in the death of affected boys by early adulthood. The genetic defect responsible for DMD has been known for over 25 years, yet at present there is neither cure nor effective treatment for DMD. During early disease onset, the mdx mouse has been validated as an animal model for DMD and use of this model has led to valuable but incomplete insights into the disease process. For example, immune cells are thought to be responsible for a significant portion of muscle cell death in the mdx mouse; however, the role and time course of the immune response in the dystrophic process have not been well described. In this paper we constructed a simple mathematical model to investigate the role of the immune response in muscle degeneration and subsequent regeneration in the mdx mouse model of Duchenne muscular dystrophy. Our model suggests that the immune response contributes substantially to the muscle degeneration and regeneration processes. Furthermore, the analysis of the model predicts that the immune system response oscillates throughout the life of the mice, and the damaged fibers are never completely cleared. PMID:25013809

  6. Myeloid deletion of SIRT1 suppresses collagen-induced arthritis in mice by modulating dendritic cell maturation

    PubMed Central

    Woo, Seong Ji; Lee, Sang-Myeong; Lim, Hye Song; Hah, Young-Sool; Jung, In Duk; Park, Yeong-Min; Kim, Hyun-Ok; Cheon, Yun-Hong; Jeon, Min-Gyu; Jang, Kyu Yun; Kim, Kyeong Min; Park, Byung-Hyun; Lee, Sang-Il

    2016-01-01

    The type III histone deacetylase silent information regulator 1 (SIRT1) is an enzyme that is critical for the modulation of immune and inflammatory responses. However, the data on its role in rheumatoid arthritis (RA) are limited and controversial. To better understand how SIRT1 regulates adaptive immune responses in RA, we evaluated collagen-induced arthritis (CIA) in myeloid cell-specific SIRT1 knockout (mSIRT1 KO) and wild-type (WT) mice. Arthritis severity was gauged on the basis of clinical, radiographic and pathologic scores. Compared with their WT counterparts, the mSIRT1 KO mice exhibited less severe arthritis, which was less destructive to the joints. The expression levels of inflammatory cytokines, matrix metalloproteinases and ROR-γT were also reduced in the mSIRT1 KO mice compared with the WT mice and were paralleled by reductions in the numbers of Th1 and Th17 cells and CD80- or CD86-positive dendritic cells (DCs). In addition, impaired DC maturation and decreases in the Th1/Th17 immune response were observed in the mSIRT1 KO mice. T-cell proliferation was also investigated in co-cultures with antigen-pulsed DCs. In the co-cultures, the DCs from the mSIRT1 KO mice showed decreases in T-cell proliferation and the Th1/Th17 immune response. In this study, myeloid cell-specific deletion of SIRT1 appeared to suppress CIA by modulating DC maturation. Thus, a careful investigation of DC-specific SIRT1 downregulation is needed to gauge the therapeutic utility of agents targeting SIRT1 in RA. PMID:26987484

  7. In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.

    PubMed

    Kapsa, R; Quigley, A; Lynch, G S; Steeper, K; Kornberg, A J; Gregorevic, P; Austin, L; Byrne, E

    2001-04-10

    Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans. PMID:11426463

  8. Early life stress accelerates behavioral and neural maturation of the hippocampus in male mice.

    PubMed

    K G, Bath; G, Manzano-Nieves; H, Goodwill

    2016-06-01

    Early life stress (ELS) increases the risk for later cognitive and emotional dysfunction. ELS is known to truncate neural development through effects on suppressing cell birth, increasing cell death, and altering neuronal morphology, effects that have been associated with behavioral profiles indicative of precocious maturation. However, how earlier silencing of growth drives accelerated behavioral maturation has remained puzzling. Here, we test the novel hypothesis that, ELS drives a switch from growth to maturation to accelerate neural and behavioral development. To test this, we used a mouse model of ELS, fragmented maternal care, and a cross-sectional dense sampling approach focusing on hippocampus and measured effects of ELS on the ontogeny of behavioral development and biomarkers of neural maturation. Consistent with previous work, ELS was associated with an earlier developmental decline in expression of markers of cell proliferation (Ki-67) and differentiation (doublecortin). However, ELS also led to a precocious arrival of Parvalbumin-positive cells, led to an earlier switch in NMDA receptor subunit expression (marker of synaptic maturity), and was associated with an earlier rise in myelin basic protein expression (key component of the myelin sheath). In addition, in a contextual fear-conditioning task, ELS accelerated the timed developmental suppression of contextual fear. Together, these data provide support for the hypothesis that ELS serves to switch neurodevelopment from processes of growth to maturation and promotes accelerated development of some forms of emotional learning. PMID:27155103

  9. The Nuclear Maturation and Embryo Development of Mice Germinal Vesicle Oocytes with and without Cumulus Cell after Vitrification

    PubMed Central

    Nikseresht, Mohsen; Toori, Mehdi Akbartabar; Rasti, Tahere; Kashani, Iraj Ragerdi

    2015-01-01

    Background: Cryobiology is an essential tool in assisted reproductive technology. Research in this area focuses on the possibility of restoring fertility in women with reproductive problems or after cancer treatments. Aim: The purpose of this study was to evaluate viability of oocytes, In vitro maturation and embryo development in vitrified germinal vesicle oocytes with and without cumulus cell after single and stepwise vitrification procedure. Materials and Methods: Germinal vesicle oocytes with or without cumulus cells were obtained from 4 weeks old female mice 48h after intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin (PMSG). For vitrification collected oocytes vitrification were exposed to cryoprotectant, which was composed of 30% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After exposure to cryoprotectant and immerged in liquid nitrogen, the oocytes were thawed and washed in medium TCM199 two times. Then the oocytes transferred to IVM medium for maturation and embryo development to blastocyst. Results: The oocytes survival rates after vitrifying-warming, maturation rate, the capacity of fertilization and embryonic development to blastocyst were examined in vitro. The oocytes survival, maturation to MII, fertilization developmental rate in the step-wise exposure and with cumulus cell was significantly higher (p<0.05) as compared with corresponding rate in the single step procedure without cumulus cell. Conclusion: The results of present study indicated that oocytes vitrified with cumulus cells and stepwise procedure had positive effect on maturation and developmental rate to blastocyst than oocytes without cumulus cell and single step procedure. PMID:25737969

  10. Sexual maturation and fertility of male and female mice exposed prenatally and postnatally to trivalent and hexavalent chromium compounds.

    PubMed

    Al-Hamood, M H; Elbetieha, A; Bataineh, H

    1998-01-01

    The reproductive toxicity of trivalent and hexavalent chromium compounds was investigated in male and female mice exposed to 1000 ppm chromium chloride and potassium dichromate via their mother during gestational and lactational periods. Fertility was reduced in male offspring exposed to either trivalent or hexavalent chromium compounds. Body weights and weights of testes, seminal vesicles and preputial glands were reduced in trivalent-exposed male offspring. The exposure of female mice offspring to trivalent and hexavalent chromium compounds delayed sexual maturation. Fertility was reduced in female offspring exposed to either trivalent or hexavalent chromium compounds. The exposure of female mice to hexavalent chromium compound reduced the number of implantations and viable fetuses respectively. Body weight and weights of ovaries and uteri were reduced in trivalent-exposed female offspring. The results indicate that under our experimental conditions, the exposure of male and female mice offspring to either trivalent or hexavalent chromium compounds during gestational and lactational periods impair reproductive functions and fertility in adulthood. PMID:9801270

  11. Reduced Connexin26 in the Mature Cochlea Increases Susceptibility to Noise-Induced Hearing Loss in Mice

    PubMed Central

    Zhou, Xing-Xing; Chen, Sen; Xie, Le; Ji, Yu-Zi; Wu, Xia; Wang, Wen-Wen; Yang, Qi; Yu, Jin-Tao; Sun, Yu; Lin, Xi; Kong, Wei-Jia

    2016-01-01

    Connexin26 (Cx26, encoded by GJB2) mutations are the most common cause of non-syndromic deafness. GJB2 is thought to be involved in noise-induced hearing loss (NIHL). However, the role of Cx26 in NIHL is still obscure. To explore the association between Cx26 and NIHL, we established a Cx26 knockdown (KD) mouse model by conditional knockdown of Cx26 at postnatal day 18 (P18), and then we observed the auditory threshold and morphologic changes in these mice with or without noise exposure. The Cx26 KD mice did not exhibit substantial hearing loss and hair cell degeneration, while the Cx26 KD mice with acoustic trauma experienced higher hearing loss than simple noise exposure siblings and nearly had no recovery. Additionally, extensive outer hair cell loss and more severe destruction of the basal organ of Corti were observed in Cx26 KD mice after noise exposure. These data indicate that reduced Cx26 expression in the mature mouse cochlea may increase susceptibility to noise-induced hearing loss and facilitate the cell degeneration in the organ of Corti. PMID:26927086

  12. In Vitro Maturation of Cumulus-Oocyte Complexes for Efficient Isolation of Oocytes from Outbred Deer Mice

    PubMed Central

    Choi, Jung Kyu; He, Xiaoming

    2013-01-01

    Background The outbred (as with humans) deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ∼5 oocytes per animal can be obtained so far. Objective The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM) of cumulus-oocyte complexes (COCs). Methods Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII) oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF) and embryo development. Results Less than ∼5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.3±2.9 oocytes per animal by IVM (16.0±2.5) and superovulation (4.3±1.3) in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells. Significance We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research. PMID:23457518

  13. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    SciTech Connect

    Ryu, Min Sook; Woo, Min-Yeong; Kwon, Daeho; Hong, Allen E.; Song, Kye Yong; Park, Sun; Lim, In Kyoung

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.

  14. Topical Application of Insulin Accelerates Vessel Maturation of Wounds by Regulating Angiopoietin-1 in Diabetic Mice.

    PubMed

    Li, Chaofei; Yu, Tianyi; Liu, Yan; Chen, Xuelian; Zhang, Xiong

    2015-12-01

    Reestablishment of the structural and functional microvasculature would be beneficial to promote healing of diabetic wounds. We explored the role of insulin application on microvascular maturation of diabetic wounds to determine whether it is associated with insulin-induced wound healing. We adopted the multiple injections of streptozotocin (STZ) to establish a diabetic animal model. The effect of insulin on microvessel formation, especially the effect of insulin on microvascular maturation was observed by transmission electron microscopy and laser scanning confocal microscopy. The pivotal protein regulated by insulin during healing processes was explored by tropical application neutralizing antibodies to these proteins; the specific protein was further confirmed using immunoblotting. On days 7 and 11, the blood vessel in insulin-treated wounds was surrounded by more α-smooth muscle actin (α-SMA) expressing cells. The blockage of angiopoietin-1 (Ang-1), but not angiopoietin-2 (Ang-2) or platelet-derived growth factor-B (PDGF-B), resulted in reduced maturation of newly formed blood vessels despite the presence of insulin in vivo. Further analysis showed that insulin induced an increased expression of Ang-1. The blood vessels in insulin-treated wounds showing advanced coverage of pericytes and reconstruction of new vascular basement membrane suggest that insulin is a potent accelerator of microvascular maturation, which may be involved in the mechanisms of insulin-induced wound healing. PMID:26349856

  15. Structural and Functional Maturation of Distal Femoral Cartilage and Bone during Postnatal Development and Growth in Humans and Mice

    PubMed Central

    Chan, Elaine F.; Harjanto, Ricky; Asahara, Hiroshi; Inoue, Nozomu; Masuda, Koichi; Bugbee, William D.; Firestein, Gary S.; Hosalkar, Harish S.; Lotz, Martin K.; Sah, Robert L.

    2012-01-01

    SYNOPSIS Introduction The size and shape of joints markedly affect their biomechanical properties, but the macroscopic 3-dimensional (3-D) mechanism and extent of cartilage and joint maturation during normal growth are largely unknown. Aims The purposes of this study were to qualitatively illustrate the development of the cartilage-bone interface in the knee during postnatal growth in humans and C57BL/6 wild-type mice, and to quantitatively define the 3-D shape using statistical shape modeling as well as to assess growth strain rates in the mouse distal femur. Methods Clinical computed tomography (CT) scans of asymptomatic knees (0.3–0.6mm in-plane resolution, 0.63mm slice thickness) were obtained from six patients between 4 to 12 years old. Micro-CT scans of mouse knees (9μm isotropic resolution) were from twenty-one mice between 12 to 120 days postnatal. Human and mouse images were compared qualitatively with 2-D images and 3-D reconstructions. Mouse femora shape parameters were determined with statistical shape modeling, and strain rates and directions during growth were mapped. Results The attainment of cartilage-bone interface shape of the distal femur and proximal tibia were qualitatively similar in humans and mice, with marked differences in growth plate morphology. Mouse distal femur shape was described by 11 independent parameters that accounted for >90% of total shape variation during growth. Each shape parameter described changes in specific anatomical regions of the distal femur and varied with age. Shape parameters and strains in the medial and lateral condyles, as well as intercondylar notch, varied greatly between postnatal days 16 to 30. Directions of growth strain across ages corresponded well with the appearance of anatomical landmarks within the distal femur. Conclusion Accurate quantification of the cartilage-bone interface geometry is imperative for furthering the understanding of the macroscopic mechanisms of cartilage maturation and

  16. Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine

    PubMed Central

    De Luca, Annamaria; Pierno, Sabata; Liantonio, Antonella; Cetrone, Michela; Camerino, Claudia; Simonetti, Simonetta; Papadia, Francesco; Camerino, Diana Conte

    2001-01-01

    No clear data is available about functional alterations in the calcium-dependent excitation-contraction (e-c) coupling mechanism of dystrophin-deficient muscle of mdx mice. By means of the intracellular microelectrode ‘point' voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8–12 weeks, during the active regeneration of hind limb muscles, and 6–8 months, when regeneration is complete. The EDL muscle fibres of 8–12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction- and relaxation-related calcium movements) with respect to control mice of 6–8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without effect on 6–8-month-old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse. HPLC determination of taurine content at 6–8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid. The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid. PMID:11226135

  17. Ovariectomy in mature mice does not increase food intake, but increases adiposity and adipose tissue inflammation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Menopause, characterized by reduced estrogen (E2), is associated with increased adiposity and metabolic pathology. Molecular mechanisms underlying this association between low E2 status and metabolic disease are not fully elucidated. When mice are fed a high fat diet (HFD) to induce obesity and diab...

  18. Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.

    PubMed

    Foltz, Steven J; Modi, Jill N; Melick, Garrett A; Abousaud, Marin I; Luan, Junna; Fortunato, Marisa J; Beedle, Aaron M

    2016-01-01

    Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury. PMID:26751696

  19. Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice

    PubMed Central

    Foltz, Steven J.; Modi, Jill N.; Melick, Garrett A.; Abousaud, Marin I.; Luan, Junna; Fortunato, Marisa J.; Beedle, Aaron M.

    2016-01-01

    Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury. PMID:26751696

  20. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice.

    PubMed

    Gong, Guohua; Song, Moshi; Csordas, Gyorgy; Kelly, Daniel P; Matkovich, Scot J; Dorn, Gerald W

    2015-12-01

    In developing hearts, changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here, we found that PINK1-Mfn2-Parkin-mediated mitophagy directs this metabolic transformation in mouse hearts. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five-week-old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts. PMID:26785495

  1. Defective T-lymphocyte migration to muscles in dystrophin-deficient mice.

    PubMed

    Cascabulho, Cynthia M; Bani Corrêa, Cristiane; Cotta-de-Almeida, Vinícius; Henriques-Pons, Andrea

    2012-08-01

    Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3500 males, is caused by mutations in the dystrophin gene. DMD leads to degeneration of skeletal and cardiac muscles and to chronic inflammation. The mdx/mdx mouse has been widely used to study DMD; this model mimics most characteristics of the disease, including low numbers of T cells in damaged muscles. In this study, we aimed to assess migration of T cells to the heart and to identify any alterations in adhesion molecules that could possibly modulate this process. In 6-week-old mdx/mdx mice, blood leukocytes, including T cells, were CD62L(+), but by 12 weeks of age down-modulation was evident, with only approximately 40% of T cells retaining this molecule. Our in vitro and in vivo results point to a P2X7-dependent shedding of CD62L (with high levels in the serum), which in 12-week-old mdx/mdx mice reduces blood T cell competence to adhere to cardiac vessels in vitro and to reach cardiac tissue in vivo, even after Trypanosoma cruzi infection, a known inducer of lymphoid myocarditis. In mdx/mdx mice treated with Brilliant Blue G, a P2X7 blocker, these blood lymphocytes retained CD62L and were capable of migrating to the heart. These results provide new insights into the mechanisms of inflammatory infiltration and immune regulation in DMD. PMID:22733008

  2. Dissociation of somatic growth, time of sexual maturity, and life expectancy by overexpression of an RGD-deficient IGFBP-2 variant in female transgenic mice.

    PubMed

    Hoeflich, Andreas; Reyer, Anja; Ohde, Daniela; Schindler, Nancy; Brenmoehl, Julia; Spitschak, Marion; Langhammer, Martina; Tuchscherer, Armin; Wirthgen, Elisa; Renner-Müller, Ingrid; Wanke, Rüdiger; Metzger, Friedrich; Bielohuby, Maximilian; Wolf, Eckhard

    2016-02-01

    Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D-mice) or mutant insulin-like growth factor-binding protein-2 (IGFBP-2) lacking the Arg-Gly-Asp (RGD) motif (E- mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (-9% and -10%) in comparison with wild-type controls (C-mice). While in D-mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C-mice, these parameters were unaltered in E-mice in spite of their reduced growth rate. These observations indicate that the RGD-domain has a major influence on the pleiotropic effects of IGFBP-2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously. PMID:26507795

  3. Prophylactic pamidronate partially protects from glucocorticoid-induced bone loss in the mdx mouse model of Duchenne muscular dystrophy.

    PubMed

    Yoon, Sung-Hee; Chen, Jinghan; Grynpas, Marc D; Mitchell, Jane

    2016-09-01

    Glucocorticoids are extensively used to treat patients with Duchenne muscular dystrophy because of their ability to delay muscle damage, prolong ambulation and extend life. However, use of glucocorticoids significantly increases bone loss, fragility and fractures. To determine if antiresorptive bisphosphonates could prevent the effects of glucocorticoids on bone quality, we used dystrophic mdx mice treated with the glucocorticoid prednisone during 8weeks of rapid bone growth from 5 to 13weeks of age and treated some mice with the bisphosphonate pamidronate during the first two weeks of prednisone administration. Prednisone reduced long bone growth, decreased cortical bone thickness and area and decreased the strength of the femurs. Pamidronate treatment protected mice from cortical bone loss but did not increase bone strength. The combination of prednisone and pamidronate inhibited remodeling of metaphyseal trabecular bone with large numbers of trabeculae containing remnants of calcified cartilage. Prednisone improved muscle strength in the mdx mice and decreased serum creatine kinase with evidence of improved muscle histology and these effects were maintained in mice treated with pamidronate. PMID:27373502

  4. The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse

    PubMed Central

    Zhang, Aiping; Uaesoontrachoon, Kitipong; Shaughnessy, Conner; Das, Jharna R.; Rayavarapu, Sree; Brown, Kristy J; Ray, Patricio E.; Nagaraju, Kanneboyina; van den Anker, John N.; Hoffman, Eric P; Hathout, Yetrib

    2015-01-01

    Phosphorodiamidate morpholino oligonucleotides (PMO) are used as a promising exon-skipping gene therapy for Duchenne Muscular Dystrophy (DMD). One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM) strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23). We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10) mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg). Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI). The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at day 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO. Chemical compounds studied in this article: Phosphorodiamidate morpholino (PubChem CID: 22140692); isoflurane (PubChem CID: 3763); formic acid (PubChem CID: 284); acetonitrile (PubChem CID: 6342); acetone (PubChem CID: 180); methanol (PubChem CID: 887) PMID:26213685

  5. The Molecular Basis of Muscular Dystrophy in the mdx Mouse: A Point Mutation

    NASA Astrophysics Data System (ADS)

    Sicinski, Piotr; Geng, Yan; Ryder-Cook, Allan S.; Barnard, Eric A.; Darlison, Mark G.; Barnard, Pene J.

    1989-06-01

    The mdx mouse is an X-linked myopathic mutant, an animal model for human Duchenne muscular dystrophy. In both mouse and man the mutations lie within the dystrophin gene, but the phenotypic differences of the disease in the two species confer much interest on the molecular basis of the mdx mutation. The complementary DNA for mouse dystrophin has been cloned, and the sequence has been used in the polymerase chain reaction to amplify normal and mdx dystrophin transcripts in the area of the mdx mutation. Sequence analysis of the amplification products showed that the mdx mouse has a single base substitution within an exon, which causes premature termination of the polypeptide chain.

  6. Andrographolide reduces cognitive impairment in young and mature AβPPswe/PS-1 mice.

    PubMed

    Serrano, Felipe G; Tapia-Rojas, Cheril; Carvajal, Francisco J; Hancke, Juan; Cerpa, Waldo; Inestrosa, Nibaldo C

    2014-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder in which the amyloid-β (Aβ) oligomers are a key factor in synaptic impairment and in spatial memory decline associated with neuronal dysfunction. This impairment includes synaptic failure associated with the loss of synaptic proteins that contribute to AD progression. Interestingly, the use of natural compounds is an emergent conceptual strategy in the search for drugs with therapeutic potentials for treating neurodegenerative disorders. In the present study, we report that andrographolide (ANDRO), which is a labdane diterpene extracted from Andrographis paniculata, increases slope of field excitatory postsynaptic potentials (fEPSP) in the CA1 region of hippocampal slices and inhibits long-term depression (LTD), protecting the long-term potentiation (LTP) against the damage induced by Aβ oligomers in vitro, most likely by inhibiting glycogen synthase kinase-3β (GSK-3β). Additionally, ANDRO prevents changes in neuropathology in two different age groups (7- and 12-month-old mice) of an AβPPswe/PS-1 Alzheimer's model. ANDRO reduces the Aβ levels, changing the ontogeny of amyloid plaques in hippocampi and cortices in 7-month-old mice, and reduces tau phosphorylation around the Aβ oligomeric species in both age groups. Additionally, we observed that ANDRO recovers spatial memory functions that correlate with protecting synaptic plasticity and synaptic proteins in two different age groups. Our results suggest that ANDRO could be used in a potential preventive therapy during AD progression. PMID:25524173

  7. Absence of Dp71 in mdx3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS

    PubMed Central

    Hernández-González, Enrique O; Mornet, Dominique; Rendon, Alvaro; Martínez-Rojas, Dalila

    2005-01-01

    Summary In muscle, the absence of dystrophin alters the dystrophin associated protein complex (DAPC), which is involved in the clustering and anchoring of signaling proteins and ion and water channels. Here we show that mice spermatozoa express only dystrophin Dp71 and utrophin Up71. The purpose of this study was to explore the effect of the absence of Dp71 on the morphology and membrane distribution of members of the DAPC, ion channels and signaling proteins of spermatozoa obtained from dystrophic mutant mdx3cv mice. Our work indicates that although the absence of Dp71 results in a dramatic decrease in β-dystroglycan, it induces membrane redistribution and an increase in the total level of a-syntrophin, voltage dependent Na+ (μ1) and K+ (Kv1.1) channels and neural nitric oxide synthase (nNOS). The short utrophin (Up71) was upregulated and redistributed in the spermatozoa of mdx3cv mice. A significant increase in abnormal flagella morphology was observed in the absence of Dp71, which was partially corrected when the plasma membrane was eliminated by detergent treatment. Our observations point to a new phenotype associated with the absence of Dp71. Abnormal flagellar structure and altered distribution of ion channels and signaling proteins may be responsible for the fertility problems of mdx3cv mice. PMID:15601658

  8. Effects of Thioglycolic Acid on in vivo Oocytes Maturation in Mice

    PubMed Central

    Ren, Xiaomei; Wang, Zhuoran

    2011-01-01

    Background Thioglycolic acid (TGA) is widely used in the hairdressing industry, which mostly caters to women. Recently, TGA has been reported to impair several organs, especially reproductive ones such as testes and ovaries. The reproductive toxicity of TGA on females has become an issue that cannot be neglected. Methodology/Principal Findings In the present work, superovulated female mice were percutaneously treated with different doses of TGA (37.81, 75.62, and 151.25 mg/kg). The mice were sacrificed to collect ovulated oocytes, whose numbers were counted and compared. Immunofluorescence-stained oocytes were observed under a confocal microscope to investigate the effects of TGA on spindle morphology, distribution of cortical granules (CGs), and parthenogenetic activation. The number of ovulated oocytes was decreased by TGA. The ovulated oocytes in the 151.25 mg/kg TGA group were significantly less than in the control and in the 37.81 mg/kg TGA groups. The ovulated oocytes in the 75.62 mg/kg TGA group were less than in the 37.81 mg/kg dose group. Abnormal spindle configuration in vivo was also induced by TGA. The spindle areas in the 75.62 and 151.25 mg/kg TGA groups were significantly larger than in the control and 37.81 mg/kg TGA groups. The parthenogenetic activation of ovulated oocytes in vitro was inhibited as well. The percentage of activated oocytes in the 75.62 and 151.25 mg/kg TGA groups was significantly lower than in the control and 37.81 mg/kg TGA groups. The percentage in the 151.25 mg/kg TGA group was also less than in the 75.62 mg/kg group. CG distribution was not affected by TGA. Conclusion Mice were percutaneously treated with TGA. Consequently, the number of ovulated oocytes decreased, abnormal spindle configurations were induced, and the parthenogenetic activation of ovulated oocytes was inhibited. CG distribution was not affected. PMID:21909408

  9. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B−/− mice

    PubMed Central

    Peixoto, Rui T.; Wang, Wengang; Croney, Donyell M.; Kozorovitskiy, Yevgenia; Sabatini, Bernardo L.

    2016-01-01

    Some autistic individuals exhibit abnormal development of the caudate nucleus and associative cortical areas, suggesting potential dysfunction of cortico-basal ganglia (BG) circuits. Using optogenetic and electrophysiological approaches in mice we identified a narrow postnatal period characterized by extensive glutamatergic synaptogenesis in striatal spiny projection neurons (SPNs) and a concomitant increase in corticostriatal circuit activity. SPNs during early development have high intrinsic excitability and respond strongly to cortical afferents despite sparse excitatory inputs. As a result, striatum and corticostriatal connectivity are highly sensitive to acute and chronic changes in cortical activity, suggesting that early imbalances in cortical function alter BG development. Indeed, a mouse model of autism with deletions in SHANK3 (Shank3B−/−) has early cortical hyperactivity, which triggers increased SPN excitatory synapse and corticostriatal hyper-connectivity. These results show a tight functional coupling between cortex and striatum during early postnatal development and suggest a potential common circuit dysfunction caused by cortical hyperactivity. PMID:26928064

  10. Accelerated Maturation of Human Stem Cell-Derived Pancreatic Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice

    PubMed Central

    Bruin, Jennifer E.; Asadi, Ali; Fox, Jessica K.; Erener, Suheda; Rezania, Alireza; Kieffer, Timothy J.

    2015-01-01

    Summary Pluripotent human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19–21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature β cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation. PMID:26677767

  11. Overview of MDX-A System for Medical Diagnosis

    PubMed Central

    Mittal, S.; Chandrasekaran, B.; Smith, J.

    1979-01-01

    We describe the design and performance of MDX, an experimental medical diagnosis system, which currently diagnoses in the syndrome called Cholestasis. The needed medical knowledge is represented in a scheme called conceptual structures, which can be viewed as a collection of conceptual experts interacting according to certain well-defined principles. MDX has three components: the diagnostic system, a patient data base and a radiology consultant. We describe these components, the inter-expert communication system and the query language used by these components. The system is illustrated by means of its performance on a real case.

  12. Schistosoma mansoni: interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

    SciTech Connect

    James, E.R.; Dobinson, A.R.

    1984-06-01

    Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of 60Co irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CI). Development into adult worms was prevented with 4 krad for IC schistosomula, but for CI schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed.

  13. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B(-/-) mice.

    PubMed

    Peixoto, Rui T; Wang, Wengang; Croney, Donyell M; Kozorovitskiy, Yevgenia; Sabatini, Bernardo L

    2016-05-01

    Some autistic individuals exhibit abnormal development of the caudate nucleus and associative cortical areas, suggesting potential dysfunction of cortico-basal ganglia (BG) circuits. Using optogenetic and electrophysiological approaches in mice, we identified a narrow postnatal period that is characterized by extensive glutamatergic synaptogenesis in striatal spiny projection neurons (SPNs) and a concomitant increase in corticostriatal circuit activity. SPNs during early development have high intrinsic excitability and respond strongly to cortical afferents despite sparse excitatory inputs. As a result, striatum and corticostriatal connectivity are highly sensitive to acute and chronic changes in cortical activity, suggesting that early imbalances in cortical function alter BG development. Indeed, a mouse model of autism with deletions in Shank3 (Shank3B(-/-)) shows early cortical hyperactivity, which triggers increased SPN excitatory synapse and corticostriatal hyperconnectivity. These results indicate that there is a tight functional coupling between cortex and striatum during early postnatal development and suggest a potential common circuit dysfunction that is caused by cortical hyperactivity. PMID:26928064

  14. Phthalate esters affect maturation and function of primate testis tissue ectopically grafted in mice

    PubMed Central

    Rodriguez-Sosa, Jose R; Bondareva, Alla; Tang, Lin; Avelar, Gleide F.; Coyle, Krysta M.; Modelski, Mark; Alpaugh, Whitney; Conley, Alan; Wynne-Edwards, Katherine; França, Luiz R; Meyers, Stuart; Dobrinski, Ina

    2014-01-01

    Di-n-Butyl (DBP) and Di-(2-EthylHexyl) (DEHP) phthalates can leach from daily-use products resulting in environmental exposure. In male rodents, phthalate exposure results in reproductive effects. To evaluate effects on the immature primate testis, testis fragments from 6-month-old rhesus macaques were grafted subcutaneously to immune-deficient mice, which were exposed to 0, 10, or 500 mg/kg of DBP or DEHP for 14 weeks or 28 weeks (DBP only). DBP exposure reduced the expression of key steroidogenic genes, indicating that Leydig cell function was compromised. Exposure to 500 mg/kg impaired tubule formation and germ cell differentiation and reduced numbers of spermatogonia. Exposure to 10 mg/kg did not affect development, but reduced Sertoli cell number and resulted in increased expression of inhibin B. Exposure to DEHP for 14 week also affected steroidogenic genes expression. Therefore, long-term exposure to phthalate esters affected development and function of the primate testis in a time and dosage dependent manner. PMID:25450860

  15. Early right ventricular fibrosis and reduction in biventricular cardiac reserve in the dystrophin-deficient mdx heart.

    PubMed

    Meyers, Tatyana A; Townsend, DeWayne

    2015-02-15

    Duchenne muscular dystrophy (DMD) is a progressive disease of striated muscle deterioration. Respiratory and cardiac muscle dysfunction are particularly clinically relevant because they result in the leading causes of death in DMD patients. Despite the clinical and physiological significance of these systems, little has been done to understand the cardiorespiratory interaction in DMD. We show here that prior to the onset of global cardiac dysfunction, dystrophin-deficient mdx mice have increased cardiac fibrosis with the right ventricle being particularly affected. Using a novel biventricular cardiac catheterization technique coupled with cardiac stress testing, we demonstrate that both the right and left ventricles have significant reductions in both systolic and diastolic function in response to dobutamine. Unstimulated cardiac function is relatively normal except for a significant reduction in the ventricular pressure transient duration compared with controls. These biventricular analyses also reveal the absence of a dobutamine-induced increase in isovolumic relaxation in the right ventricle of control hearts. Simultaneous assessment of biventricular pressure demonstrates a dobutamine-dependent enhancement of coupling between the ventricles in control mice, which is absent in mdx mice. Furthermore, studies probing the passive-extension properties of the left ventricle demonstrate that the mdx heart is significantly more compliant compared with age-matched C57BL/10 hearts, which have an age-dependent stiffening that is completely absent from dystrophic hearts. These new results indicate that right ventricular fibrosis is an early indicator of the development of dystrophic cardiomyopathy, suggesting a mechanism by which respiratory insufficiency may accelerate the development of heart failure in DMD. PMID:25485898

  16. Efficacy of Cyclooctadepsipeptides and Aminophenylamidines against Larval, Immature and Mature Adult Stages of a Parasitologically Characterized Trichurosis Model in Mice

    PubMed Central

    Kulke, Daniel; Krücken, Jürgen; Harder, Achim; von Samson-Himmelstjerna, Georg

    2014-01-01

    Background The genus Trichuris includes parasites of major relevance in veterinary and human medicine. Despite serious economic losses and enormous impact on public health, treatment options against whipworms are very limited. Additionally, there is an obvious lack of appropriately characterized experimental infection models. Therefore, a detailed parasitological characterization of a Trichuris muris isolate was performed in C57BL/10 mice. Subsequently, the in vivo efficacies of the aminophenylamidines amidantel, deacylated amidantel (dAMD) and tribendimidine as well as the cyclooctadepsipeptides emodepside and in particular PF1022A were analyzed. This was performed using various administration routes and treatment schemes targeting histotropic and further developed larval as well as immature and mature adult stages. Methodology/Principal Findings Duration of prepatent period, time-dependent localization of larvae during period of prepatency as well as the duration of patency of the infection were determined before drugs were tested in the characterized trichurosis model. Amidantel showed no effect against mature adult T. muris. Tribendimidine showed significantly higher potency than dAMD after oral treatments (ED50 values of 6.5 vs. 15.1 mg/kg). However, the opposite was found for intraperitoneal treatments (ED50 values of 15.3 vs. 8.3 mg/kg). When emodepside and PF1022A were compared, the latter was significantly less effective against mature adults following intraperitoneal (ED50 values of 6.1 vs. 55.7 mg/kg) or subcutaneous (ED50 values of 15.2 vs. 225.7 mg/kg) administration. Only minimal differences were observed following oral administration (ED50 values of 2.7 vs. 5.2 mg/kg). Triple and most single oral doses with moderate to high dosages of PF1022A showed complete efficacy against histotropic second stage larvae (3×100 mg/kg or 1×250 mg/kg), further developed larvae (3×10 mg/kg or 1×100 mg/kg) and immature adults (3×10 mg/kg or 1×100 mg

  17. Coronary adventitial cells are linked to perivascular cardiac fibrosis via TGFβ1 signaling in the mdx mouse model of Duchenne Muscular Dystrophy

    PubMed Central

    Ieronimakis, Nicholas; Hays, Aislinn L.; Janebodin, Kajohnkiart; Mahoney, William M.; Duffield, Jeremy S.; Majesky, Mark W.; Reyes, Morayma

    2013-01-01

    In Duchenne Muscular Dystrophy (DMD), progressive accumulation of cardiac fibrosis promotes heart failure. While the cellular origins of fibrosis in DMD hearts remain enigmatic, fibrotic tissue conspicuously forms near the coronary adventitia. Therefore, we sought to characterize the role of coronary adventitial cells in the formation of perivascular fibrosis. Utilizing the mdx model of DMD, we have identified a population of Sca1+, PDGFRα+, CD31−, CD45− coronary adventitial cells responsible for perivascular fibrosis. Histopathology of dystrophic hearts revealed Sca1+ cells extend from the adventitia and occupy regions of perivascular fibrosis. The number of Sca1+ adventitial cells increased two-fold in fibrotic mdx hearts vs. age matched wild-type hearts. Moreover, relative to Sca1−, PDGFRα+, CD31−, CD45− cells and endothelial cells, Sca1+ adventitial cells FACS-sorted from mdx hearts expressed the highest level of Collagen1α1 and 3α1, Connective tissue growth factor, and Tgfβr1 transcripts. Surprisingly, mdx endothelial cells expressed the greatest level of the Tgfβ1 ligand. Utilizing Collagen1α1-GFP reporter mice, we confirmed that the majority of Sca1+ adventitial cells expressed type I collagen, an abundant component of cardiac fibrosis, in both wt (71% ±4.1) and mdx (77% ±3.5) hearts. In contrast, GFP+ interstitial fibroblasts were PDGFRα+ but negative for Sca1. Treatment of cultured Collagen1α1-GFP+ adventitial cells with TGFβ1 resulted in increased collagen synthesis, whereas pharmacological inhibition of TGFβR1 signaling reduced the fibrotic response. Therefore, perivascular cardiac fibrosis by coronary adventitial cells may be mediated by TGFβ1 signaling. Our results implicate coronary endothelial cells in mediating cardiac fibrosis via transmural TGFβ signaling, and suggest that the coronary adventitia is a promising target for developing novel anti-fibrotic therapies. PMID:23911435

  18. Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

    PubMed

    Seki, Takao; Yamamoto, Mami; Taguchi, Yuu; Miyauchi, Maki; Akiyama, Nobuko; Yamaguchi, Noritaka; Gohda, Jin; Akiyama, Taishin; Inoue, Jun-ichiro

    2015-12-01

    RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway. PMID:26115685

  19. Targeted gene correction in the mdx mouse using short DNA fragments: towards application with bone marrow-derived cells for autologous remodeling of dystrophic muscle.

    PubMed

    Kapsa, R M; Quigley, A F; Vadolas, J; Steeper, K; Ioannou, P A; Byrne, E; Kornberg, A J

    2002-06-01

    In muscle, mutant genes can be targeted and corrected directly by intramuscular (i.m.) injection of corrective DNA, or by ex vivo delivery of DNA to myogenic cells, followed by cell transplantation. Short fragment homologous replacement (SFHR) has been used to repair the exon 23 nonsense transition at the Xp21.1 dys locus in cultured cells and also, directly in tibialis anterior from male mdx mice. Whilst mdx dys locus correction can be achieved in up to 20% of cells in culture, much lower efficiency is evident by i.m. injection. The major consideration for application of targeted gene correction to muscle is delivery throughout relevant tissues. Systemically injected bone marrow (BM)-derived cells from wt C57BL/10 ScSn mice are known to remodel mdx muscle when injected into the systemic route. Provided that non muscle-derived cell types most capable of muscle remodeling activity can be more specifically identified, isolated and expanded, cell therapy seems presently the most favorable vehicle by which to deliver gene correction throughout muscle tissues. Using wt bone marrow as a model, this study investigates systemic application of bone marrow-derived cells as potential vehicles to deliver corrected (ie wt) dys locus to dystrophic muscle. Intravenous (i.v.) and intraperitoneal (i.p.) injections of wt BM were given to lethally and sub-lethally irradiated mdx mice. Despite both i.v. and surviving i.p. groups containing wt dys loci in 100% and less than 1% of peripheral blood nuclei, respectively, both groups displayed equivalent levels of wt dys transcript in muscle RNA. These results suggest that the muscle remodeling activity observed in systemically injected BM cells is not likely to be found in the hemopoietic fraction. PMID:12032690

  20. Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts.

    PubMed

    Moisset, P A; Gagnon, Y; Karpati, G; Tremblay, J P

    1998-10-01

    Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppressive drugs. To determine the conditions to be used in this kind of approach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI or 0.01-1000) with an adenoviral vector containing a CMV promoter/enhancer driven 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for transgene expression. In these cultures, dystrophin mRNA was found to be proportionate with increasing MOI. Primary myoblast cultures derived from transgenic mdx mice expressing beta-Gal under a muscle-specific promoter and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibialis muscles of SCID mice. Ten and 24 days after transplantation, numerous muscle fibers expressing both human dystrophin and beta-Gal were detected throughout the mouse muscles by immunohistochemistry using an antibody specific for human dystrophin. The presence of the human mini-dystrophin mRNA was also detected by RT-PCR. These results demonstrate that three essential conditions in autologous myoblast transplantation can be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle fibers; and (3) the high expression of the dystrophin transgene in situ. Furthermore, this article provides a novel RT-PCR-based technique to quantify the human dystrophin minigene expression in vitro and in vivo. PMID:9930339

  1. Transgenic mice overexpressing glia maturation factor-β, an oxidative stress inducible gene, show premature aging due to Zmpste24 down-regulation

    PubMed Central

    Hanai, Jun-ichi; Takenaka, Masaru

    2015-01-01

    Glia Maturation Factor-β (GMF), a brain specific protein, is induced by proteinuria in renal tubules. Ectopic GMF overexpression causes apoptosis in vitro via cellular vulnerability to oxidative stress. In order to examine the roles of GMF in non-brain tissue, we constructed transgenic mice overexpressing GMF (GMF-TG). The GMF-TG mice exhibited appearance phenotypes associated with premature aging. The GMF-TG mice also demonstrated short lifespans and reduced hair regrowth, suggesting an accelerated aging process. The production of an abnormal lamin A, a nuclear envelope protein, plays a causal role in both normal aging and accelerated aging diseases, known as laminopathies. Importantly, we identified the abnormal lamin A (prelamin A), accompanied by a down-regulation of a lamin A processing enzyme (Zmpste24) in the kidney of the GMF-TG mice. The GMF-TG mice showed accelerated aging in the kidney, compared with wild-type mice, showing increased TGF-β1, CTGF gene and serum creatinine. The gene expression of p21/waf1 was increased at an earlier stage of life, at 10 weeks, which was in turn down-regulated at a later stage, at 60 weeks. In conclusion, we propose that GMF-TG mice might be a novel mouse model of accelerated aging, due to the abnormal lamin A. PMID:26232943

  2. Transgenic mice overexpressing glia maturation factor-β, an oxidative stress inducible gene, show premature aging due to Zmpste24 down-regulation.

    PubMed

    Imai, Rika; Asai, Kanae; Hanai, Jun-ichi; Takenaka, Masaru

    2015-07-01

    Glia Maturation Factor-β (GMF), a brain specific protein, is induced by proteinuria in renal tubules. Ectopic GMF overexpression causes apoptosisin vitro via cellular vulnerability to oxidative stress. In order to examine the roles of GMF in non-brain tissue, we constructed transgenic mice overexpressing GMF (GMF-TG). The GMF-TG mice exhibited appearance phenotypes associated with premature aging. The GMF-TG mice also demonstrated short lifespans and reduced hair regrowth, suggesting an accelerated aging process. The production of an abnormal lamin A, a nuclear envelope protein, plays a causal role in both normal aging and accelerated aging diseases, known as laminopathies. Importantly, we identified the abnormal lamin A (prelamin A), accompanied by a down-regulation of a lamin A processing enzyme (Zmpste24) in the kidney of the GMF-TG mice. The GMF-TG mice showed accelerated aging in the kidney, compared with wild-type mice, showing increased TGF-β1, CTGF gene and serum creatinine. The gene expression of p21/waf1 was increased at an earlier stage of life, at 10 weeks, which was in turn down-regulated at a later stage, at 60 weeks. In conclusion, we propose that GMF-TG mice might be a novel mouse model of accelerated aging, due to the abnormal lamin A. PMID:26232943

  3. Deletion of Galgt2 (B4Galnt2) reduces muscle growth in response to acute injury and increases muscle inflammation and pathology in dystrophin-deficient mice.

    PubMed

    Xu, Rui; Singhal, Neha; Serinagaoglu, Yelda; Chandrasekharan, Kumaran; Joshi, Mandar; Bauer, John A; Janssen, Paulus M L; Martin, Paul T

    2015-10-01

    Transgenic overexpression of Galgt2 (official name B4Galnt2) in skeletal muscle stimulates the glycosylation of α dystroglycan (αDG) and the up-regulation of laminin α2 and dystrophin surrogates known to inhibit muscle pathology in mouse models of congenital muscular dystrophy 1A and Duchenne muscular dystrophy. Skeletal muscle Galgt2 gene expression is also normally increased in the mdx mouse model of Duchenne muscular dystrophy compared with the wild-type mice. To assess whether this increased endogenous Galgt2 expression could affect disease, we quantified muscular dystrophy measures in mdx mice deleted for Galgt2 (Galgt2(-/-)mdx). Galgt2(-/-) mdx mice had increased heart and skeletal muscle pathology and inflammation, and also worsened cardiac function, relative to age-matched mdx mice. Deletion of Galgt2 in wild-type mice also slowed skeletal muscle growth in response to acute muscle injury. In each instance where Galgt2 expression was elevated (developing muscle, regenerating muscle, and dystrophic muscle), Galgt2-dependent glycosylation of αDG was also increased. Overexpression of Galgt2 failed to inhibit skeletal muscle pathology in dystroglycan-deficient muscles, in contrast to previous studies in dystrophin-deficient mdx muscles. This study demonstrates that Galgt2 gene expression and glycosylation of αDG are dynamically regulated in muscle and that endogenous Galgt2 gene expression can ameliorate the extent of muscle pathology, inflammation, and dysfunction in mdx mice. PMID:26435413

  4. Self-Improvement of Keratinocyte Differentiation Defects During Skin Maturation in ABCA12-Deficient Harlequin Ichthyosis Model Mice

    PubMed Central

    Yanagi, Teruki; Akiyama, Masashi; Nishihara, Hiroshi; Ishikawa, Junko; Sakai, Kaori; Miyamura, Yuki; Naoe, Ayano; Kitahara, Takashi; Tanaka, Shinya; Shimizu, Hiroshi

    2010-01-01

    Harlequin ichthyosis (HI) is caused by loss-of-function mutations in the keratinocyte lipid transporter ABCA12. The patients often die in the first 1 or 2 weeks of life, although HI survivors’ phenotypes improve within several weeks after birth. In order to clarify the mechanisms of phenotypic recovery, we studied grafted skin and keratinocytes from Abca12-disrupted (Abca12−/−) mice showing abnormal lipid transport. Abca12−/− neonatal epidermis showed significantly reduced total ceramide amounts and aberrant ceramide composition. Immunofluorescence and immunoblotting of Abca12−/− neonatal epidermis revealed defective profilaggrin/filaggrin conversion and reduced protein expression of the differentiation-specific molecules, loricrin, kallikrein 5, and transglutaminase 1, although their mRNA expression was up-regulated. In contrast, Abca12−/− skin grafts kept in a dry environment exhibited dramatic improvements in all these abnormalities. Increased transepidermal water loss, a parameter representing barrier defect, was remarkably decreased in grafted Abca12−/− skin. Ten-passage sub-cultured Abca12−/− keratinocytes showed restoration of intact ceramide distribution, differentiation-specific protein expression and profilaggrin/filaggrin conversion, which were defective in primary-cultures. Using cDNA microarray analysis, lipid transporters including four ATP-binding cassette transporters were up-regulated after sub-culture of Abca12−/− keratinocytes compared with primary-culture. These results indicate that disrupted keratinocyte differentiation during the fetal development is involved in the pathomechanism of HI and, during maturation, Abca12−/− epidermal keratinocytes regain normal differentiation processes. This restoration may account for the skin phenotype improvement observed in HI survivors. PMID:20489143

  5. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

    PubMed Central

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  6. Partial restoration of cardiac function with ΔPDZ nNOS in aged mdx model of Duchenne cardiomyopathy

    PubMed Central

    Lai, Yi; Zhao, Junling; Yue, Yongping; Wasala, Nalinda B.; Duan, Dongsheng

    2014-01-01

    Transgenic gene deletion/over-expression studies have established the cardioprotective role of neuronal nitric oxide synthase (nNOS). However, it remains unclear whether nNOS-mediated heart protection can be translated to gene therapy. In this study, we generated an adeno-associated virus (AAV) nNOS vector and tested its therapeutic efficacy in the aged mdx model of Duchenne cardiomyopathy. A PDZ domain-deleted nNOS gene (ΔPDZ nNOS) was packaged into tyrosine mutant AAV-9 and delivered to the heart of ∼14-month-old female mdx mice, a phenotypic model of Duchenne cardiomyopathy. Seven months later, we observed robust nNOS expression in the myocardium. Supra-physiological ΔPDZ nNOS expression significantly reduced myocardial fibrosis, inflammation and apoptosis. Importantly, electrocardiography and left ventricular hemodynamics were significantly improved in treated mice. Additional studies revealed increased phosphorylation of phospholamban and p70S6K. Collectively, we have demonstrated the therapeutic efficacy of the AAV ΔPDZ nNOS vector in a symptomatic Duchenne cardiomyopathy model. Our results suggest that the cardioprotective role of ΔPDZ nNOS is likely through reduced apoptosis, enhanced phospholamban phosphorylation and improved Akt/mTOR/p70S6K signaling. Our study has opened the door to treat Duchenne cardiomyopathy with ΔPDZ nNOS gene transfer. PMID:24463882

  7. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy.

    PubMed

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  8. Nitric oxide donors improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm.

    PubMed

    Mizunoya, Wataru; Upadhaya, Ritika; Burczynski, Frank J; Wang, Guqi; Anderson, Judy E

    2011-05-01

    In Duchenne muscular dystrophy (DMD), palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide synthase activity in dystrophic mice promotes regeneration, the outcome of two nitric oxide (NO) donor drugs, MyoNovin (M) and isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug prednisone (P) in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as controls or with an NO donor ± P. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. P decreased body weight gain, M increased quadriceps mass, and I increased heart mass. P increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO donors + P (M+P, I+P) reduced %EBD+ fibers and calcification vs. P alone. %EBD+ fibers in M+P diaphragm did not differ from control. NO donor treatment reduced proliferation and the population of c-met+ cells and accelerated fiber regeneration. Concurrent with P, NO donor treatment suppressed two important detrimental effects of P in mice, possibly by accelerating regeneration, rebalancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest that NO donors could improve current therapy for DMD. PMID:21270295

  9. Disruption of Ang-1/Tie-2 Signaling Contributes to the Impaired Myocardial Vascular Maturation and Angiogenesis in Type II Diabetic Mice

    PubMed Central

    Chen, Jian-Xiong; Stinnett, Amanda

    2016-01-01

    Objective Microvascular insufficiency represents a major cause of end-organ failure among diabetics. The current studies were undertaken to determine whether dysregulation of the angiopoietins/Tie-2 system would result in an impairment of smooth muscle cell (SMC) recruitment and vascular maturation, which contributes to impaired angiogenesis in diabetes. Methods and Results Tie-2 expression was significantly attenuated, whereas angiopoietin-2 (Ang-2) was increased in db/db mice subjected to myocardial ischemia. Our morphological analysis showed that the number of SMC coverage area per neovessel was significantly reduced in db/db mice. This was accompanied by a significant reduction of myocardial capillary density and arteriole formation. Interestingly, Angiopoietin-1(Ang-1)–induced SMC recruitment and vessel outgrowth were severely impaired in db/db mice. Our in vitro studies further demonstrated that exposure of mouse heart endothelial cells to high glucose resulted in a significant upregulation of Ang-2 and a downregulation of Tie-2 expression. These alterations led to a significant impairment of Ang-1–induced Akt and eNOS phosphorylation, along with a remarkable impairment of Ang-1–induced endothelial cell migration and endothelial cell spheroid sprouting. Ang-1 gene transfer restored Tie-2 expression and rescued these abnormalities in diabetes. Conclusions Our findings underscore the important role of Ang-1–Tie-2 signaling in the diabetes-induced impairment of vascular maturation and angiogenesis. PMID:18556567

  10. Functional and Molecular Effects of Arginine Butyrate and Prednisone on Muscle and Heart in the mdx Mouse Model of Duchenne Muscular Dystrophy

    PubMed Central

    Guerron, Alfredo D.; Rawat, Rashmi; Sali, Arpana; Spurney, Christopher F.; Pistilli, Emidio; Cha, Hee-Jae; Pandey, Gouri S.; Gernapudi, Ramkishore; Francia, Dwight; Farajian, Viken; Escolar, Diana M.; Bossi, Laura; Becker, Magali; Zerr, Patricia; de la Porte, Sabine; Gordish-Dressman, Heather; Partridge, Terence; Hoffman, Eric P.; Nagaraju, Kanneboyina

    2010-01-01

    Background The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin. Methodology/Principal Findings In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy. Conclusions/Significance These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo

  11. Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

    PubMed

    Decrouy, A; Renaud, J M; Lunde, J A; Dickson, G; Jasmin, B J

    1998-01-01

    In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions. PMID:9536265

  12. Galectin-1 Protein Therapy Prevents Pathology and Improves Muscle Function in the mdx Mouse Model of Duchenne Muscular Dystrophy.

    PubMed

    Van Ry, Pam M; Wuebbles, Ryan D; Key, Megan; Burkin, Dean J

    2015-08-01

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by mutations in the dystrophin gene, leading to the loss of a critical component of the sarcolemmal dystrophin glycoprotein complex. Galectin-1 is a small 14 kDa protein normally found in skeletal muscle and has been shown to be a modifier of immune response, muscle repair, and apoptosis. Galectin-1 levels are elevated in the muscle of mouse and dog models of DMD. Together, these findings led us to hypothesize that Galectin-1 may serve as a modifier of disease progression in DMD. To test this hypothesis, recombinant mouse Galectin-1 was produced and used to treat myogenic cells and the mdx mouse model of DMD. Here we show that intramuscular and intraperitoneal injections of Galectin-1 into mdx mice prevented pathology and improved muscle function in skeletal muscle. These improvements were a result of enhanced sarcolemmal stability mediated by elevated utrophin and α7β1 integrin protein levels. Together our results demonstrate for the first time that Galectin-1 may serve as an exciting new protein therapeutic for the treatment of DMD. PMID:26050991

  13. Rescue of a dystrophin-like protein by exon skipping normalizes synaptic plasticity in the hippocampus of the mdx mouse.

    PubMed

    Dallérac, Glenn; Perronnet, Caroline; Chagneau, Carine; Leblanc-Veyrac, Pascale; Samson-Desvignes, Nathalie; Peltekian, Elise; Danos, Olivier; Garcia, Luis; Laroche, Serge; Billard, Jean-Marie; Vaillend, Cyrille

    2011-09-01

    Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a protein that fulfills important functions in both muscle and brain. The mdx mouse model of DMD, which also lacks dystrophin, shows a marked reduction in γ-aminobutyric acid type A (GABA(A))-receptor clustering in central inhibitory synapses and enhanced long-term potentiation (LTP) at CA3-CA1 synapses of the hippocampus. We have recently shown that U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors are able to induce skipping of the mutated exon 23 and to rescue expression of a functional dystrophin-like product both in the muscle and nervous tissue in vivo. In the brain, this rescue was accompanied by restoration of both the size and number of hippocampal GABA(A)-receptor clustering. Here, we report that 25.2±8% of re-expression two months after intrahippocampal injection of rAAV reverses the abnormally enhanced LTP phenotype at CA3-CA1 synapses of mdx mice. These results suggests that dystrophin expression indirectly influences synaptic plasticity through modulation of GABA(A)-receptor clustering and that re-expression of the otherwise deficient protein in the adult can significantly alleviate alteration of neural functions in DMD. PMID:21624465

  14. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men

    PubMed Central

    O’Hara, Laura; McInnes, Kerry; Simitsidellis, Ioannis; Morgan, Stephanie; Atanassova, Nina; Slowikowska-Hilczer, Jolanta; Kula, Krzysztof; Szarras-Czapnik, Maria; Milne, Laura; Mitchell, Rod T.; Smith, Lee B.

    2015-01-01

    Leydig cell number and function decline as men age, and low testosterone is associated with all “Western” cardio-metabolic disorders. However, whether perturbed androgen action within the adult Leydig cell lineage predisposes individuals to this late-onset degeneration remains unknown. To address this, we generated a novel mouse model in which androgen receptor (AR) is ablated from ∼75% of adult Leydig stem cell/cell progenitors, from fetal life onward (Leydig cell AR knockout mice), permitting interrogation of the specific roles of autocrine Leydig cell AR signaling through comparison to adjacent AR-retaining Leydig cells, testes from littermate controls, and to human testes, including from patients with complete androgen insensitivity syndrome (CAIS). This revealed that autocrine AR signaling is dispensable for the attainment of final Leydig cell number but is essential for Leydig cell maturation and regulation of steroidogenic enzymes in adulthood. Furthermore, these studies reveal that autocrine AR signaling in Leydig cells protects against late-onset degeneration of the seminiferous epithelium in mice and inhibits Leydig cell apoptosis in both adult mice and patients with CAIS, possibly via opposing aberrant estrogen signaling. We conclude that autocrine androgen action within Leydig cells is essential for the lifelong support of spermatogenesis and the development and lifelong health of Leydig cells.—O’Hara, L., McInnes, K., Simitsidellis, I., Morgan, S., Atanassova, N., Slowikowska-Hilczer, J., Kula, K., Szarras-Czapnik, M., Milne, L., Mitchell, R. T., Smith, L. B. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men. PMID:25404712

  15. Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice

    SciTech Connect

    Nair, Ramya; Singh, Vikram Jeet; Salian, Sujith Raj; Kalthur, Sneha Guruprasad; D'Souza, Antony Sylvan; Shetty, Pallavi K.; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

    2014-09-15

    Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20 mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5 mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20 mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome. - Highlights: • Methyl parathion induces severe cytoplasmic abnormalities in oocytes. • Inhibits nuclear maturation and spindle damage • Poor blastocyst quality and high DNA

  16. Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice.

    PubMed

    Yamada, Hiromi; Ito, Daisuke; Oki, Yoshinao; Kitagawa, Masato; Matsumoto, Taro; Watari, Tosihiro; Kano, Koichiro

    2014-11-14

    Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord. PMID:25451251

  17. Eliminating the synthesis of mature lamin A reduces disease phenotypes in mice carrying a Hutchinson-Gilford progeria syndrome allele.

    PubMed

    Yang, Shao H; Qiao, Xin; Farber, Emily; Chang, Sandy Y; Fong, Loren G; Young, Stephen G

    2008-03-14

    Hutchinson-Gilford progeria syndrome is caused by the synthesis of a mutant form of prelamin A, which is generally called progerin. Progerin is targeted to the nuclear rim, where it interferes with the integrity of the nuclear lamina, causes misshapen cell nuclei, and leads to multiple aging-like disease phenotypes. We created a gene-targeted allele yielding exclusively progerin (Lmna HG) and found that heterozygous mice (Lmna HG/+) exhibit many phenotypes of progeria. In this study, we tested the hypothesis that the phenotypes elicited by the Lmna HG allele might be modulated by compositional changes in the nuclear lamina. To explore this hypothesis, we bred mice harboring one Lmna HG allele and one Lmna LCO allele (a mutant allele that produces lamin C but no lamin A). We then compared the phenotypes of Lmna HG/LCO mice (which produce progerin and lamin C) with littermate Lmna HG/+ mice (which produce lamin A, lamin C, and progerin). Lmna HG/LCO mice exhibited improved HG/LCO fibroblasts had fewer misshapen nuclei than Lmna HG/+ fibroblasts (p < 0.0001). A likely explanation for these differences was uncovered; the amount of progerin in Lmna HG/LCO fibroblasts and tissues was lower than in Lmna HG/+ fibroblasts and tissues. These studies suggest that compositional changes in the nuclear lamina can influence both the steady-state levels of progerin and the severity of progeria-like disease phenotypes. PMID:18178963

  18. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse

    SciTech Connect

    Love, D.R.; Marsden, R.F.; Bloomfield, J.F.; Davies, K.E. ); Morris, G.E.; Ellis, J.M. ); Fairbrother, U.; Edwards, Y.H. ); Slater, C.P. ); Parry, D.J. )

    1991-04-15

    The authors have previously reported a dystrophin-related locus (DMDL for Duchenne muscular dystrophy-like) on human chromosome 6 that maps close to the dy mutation on mouse chromosome 10. Here they show that this gene is expressed in a wide range of tissues at varying levels. The transcript is particularly abundant in several human fetal tissues, including heart, placenta, and intestine. Studies with antisera raised against a DMDL fusion protein identify a 400,000 M{sub r} protein in all mouse tissues tested, including those of mdx and dy mice. Unlike the dystrophin gene, the DMDL gene transcript is not differentially spliced at the 3{prime} end in either fetal muscle or brain.

  19. Delayed and Deficient Dermal Maturation in Mice Lacking the CXCR3 ELR-Negative CXC Chemokine Receptor

    PubMed Central

    Yates, Cecelia C.; Whaley, Diana; Kulasekeran, Priya; Hancock, Wayne W.; Lu, Bao; Bodnar, Richard; Newsome, Joseph; Hebda, Patricia A.; Wells, Alan

    2007-01-01

    Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this “stop and return” process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (+/+) or knockout (−/−) mice. Even at 90 days, long after wound closure, wounds in the CXCR3−/− mice remained hypercellular and presented immature matrix components. The CXCR3−/− mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair. PMID:17600132

  20. Effect of Proton Irradiation Followed by Hindlimb Unloading on Bone in Mature Mice: A Model of Long-Duration Spaceflight

    PubMed Central

    Lloyd, Shane A.; Bandstra, Eric R.; Willey, Jeffrey S.; Riffle, Stephanie E.; Tirado-Lee, Leidamarie; Nelson, Gregory A.; Pecaut, Michael J.; Bateman, Ted A.

    2012-01-01

    Bone loss associated with microgravity unloading is well documented; however, the effects of spaceflight-relevant types and doses of radiation on the skeletal system are not well defined. In addition, the combined effect of unloading and radiation has not received much attention. In the present study, we investigated the effect of proton irradiation followed by mechanical unloading via hindlimb suspension (HLS) in mice. Sixteen-week-old female C57BL/6 mice were either exposed to 1 Gy of protons or a sham irradiation procedure (n=30/group). One day later, half of the mice in each group were subjected to four weeks of HLS or normal loading conditions. Radiation treatment alone (IRR) resulted in approximately 20% loss of trabecular bone volume fraction (BV/TV) in the tibia and femur, with no effect in the cortical bone compartment. Conversely, unloading induced substantially greater loss of both trabecular bone (60–70% loss of BV/TV) and cortical bone (approximately 20% loss of cortical bone volume) in both the tibia and femur, with corresponding decreases in cortical bone strength. Histological analyses and serum chemistry data demonstrated increased levels of osteoclast-mediated bone resorption in unloaded mice, but not IRR. HLS+IRR mice generally experienced greater loss of trabecular bone volume fraction, connectivity density, and trabecular number than either unloading or irradiation alone. Although the duration of unloading may have masked certain effects, the skeletal response to irradiation and unloading appears to be additive for certain parameters. Appropriate modeling of the environmental challenges of long duration spaceflight will allow for a better understanding of the underlying mechanisms mediating spaceflight-associated bone loss and for the development of effective countermeasures. PMID:22789684

  1. Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes

    PubMed Central

    Fanchaouy, M.; Polakova, E.; Jung, C.; Ogrodnik, J.; Shirokova, N.; Niggli, E.

    2009-01-01

    In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca2+ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca2+ influx pathways with pharmacological tools. Cytosolic Ca2+ and Na+ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca2+ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca2+ influx pathway, the Na+-Ca2+ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca2+ entry in exchange for Na+ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca2+ influx as a treatment option for dystrophy. PMID:19604578

  2. Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice

    PubMed Central

    Chiappalupi, Sara; Luca, Giovanni; Mancuso, Francesca; Madaro, Luca; Fallarino, Francesca; Nicoletti, Carmine; Calvitti, Mario; Arato, Iva; Falabella, Giulia; Salvadori, Laura; Di Meo, Antonio; Bufalari, Antonello; Giovagnoli, Stefano; Calafiore, Riccardo; Donato, Rosario; Sorci, Guglielmo

    2015-01-01

    We report data about the effects of intraperitoneal (i.p.) injection of specific pathogen-free (SPF) porcine Sertoli cells (SeC) encapsulated into clinical grade alginate-based microcapsules (SeC-MC) on muscles of chronic and presymptomatic dystrophic, mdx mice. Mdx mouse is the best characterized animal model of Duchenne muscular dystrophy (DMD), an X-linked lethal myopathy due to mutation in the gene of dystrophin, which is crucial for myofiber integrity during muscle contraction. Our data show that three weeks after i.p. injection of SeC-MC significantly reduced adipose and fibrous tissue deposition, reduced macrophage infiltrate, and reduced numbers of damaged myofibers are found in muscles of 12-month-old mdx mice, which reproduce chronic DMD conditions. Compared with muscles of mock-treated mdx mice muscles of SeC-MC-treated mice show upregulation of the dystrophin paralogue, utrophin which is localized to the periphery of myofibers. Moreover, our data show that i.p. injection of SeC-MC into presymptomatic, 2-week-old mdx mice, although not fully preventing myofiber degeneration, results in protection against myofiber necrosis and muscle inflammation. Extensive discussion of these data can be found in Ref. [1]. PMID:26759818

  3. Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice

    PubMed Central

    Chia, Ian; Grote, David; Marcotte, Michael; Batourina, Ekaterina; Mendelsohn, Cathy; Bouchard, Maxime

    2011-01-01

    Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility. PMID:21521737

  4. E-selectin ligand–1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-β

    PubMed Central

    Yang, Tao; Mendoza-Londono, Roberto; Lu, Huifang; Tao, Jianning; Li, Kaiyi; Keller, Bettina; Jiang, Ming Ming; Shah, Rina; Chen, Yuqing; Bertin, Terry K.; Engin, Feyza; Dabovic, Branka; Rifkin, Daniel B.; Hicks, John; Jamrich, Milan; Beaudet, Arthur L.; Lee, Brendan

    2010-01-01

    The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-β signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-β signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-β is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand–1 (ESL-1), acts as a negative regulator of TGF-β production by binding TGF-β precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-β by a furin-like protease, leading to reduced secretion of mature TGF-β by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-β/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-β/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-β during skeletal development and homeostasis. PMID:20530870

  5. Transient receptor potential cation channels in normal and dystrophic mdx muscle.

    PubMed

    Krüger, Jana; Kunert-Keil, Christiane; Bisping, Frederike; Brinkmeier, Heinrich

    2008-06-01

    To investigate the defective calcium regulation of dystrophin-deficient muscle fibres we studied gene expression and localization of non-voltage gated cation channels in normal and mdx mouse skeletal muscle. We found TRPC3, TRPC6, TRPV4, TRPM4 and TRPM7 to be the most abundant isoforms. Immunofluorescent staining of muscle cross-sections with antibodies against TRP proteins showed sarcolemmal localization of TRPC6 and TRPM7, both, for mdx and control. TRPV4 was found only in a fraction of fibres at the sarcolemma and around myonuclei, while TRPC3 staining revealed intracellular patches, preferentially in mdx muscle. Transcripts of low abundance coding for TRPC5, TRPA1 and TRPM1 channels were increased in mdx skeletal muscle at certain stages. The increased Ca(2+)-influx into dystrophin-deficient mdx fibres cannot be explained by increased gene expression of major TRP channels. However, a constant TRP channel expression in combination with the well described weaker Ca(2+)-handling system of mdx fibres may indicate an imbalance between Ca(2+)-influx and cellular Ca(2+)-control. PMID:18504127

  6. Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice

    PubMed Central

    Meurer, Steffen K.; Neß, Melanie; Weiskirchen, Sabine; Kim, Philipp; Tag, Carmen G.; Kauffmann, Marlies; Huber, Michael; Weiskirchen, Ralf

    2016-01-01

    Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research. PMID:27337047

  7. Pre- and postsynaptic changes in the neuromuscular junction in dystrophic mice

    PubMed Central

    Pratt, Stephen J. P.; Valencia, Ana P.; Le, Gloribel K.; Shah, Sameer B.; Lovering, Richard M.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated in mdx mice (murine model for DMD) significant morphologic alterations at the motor endplate of the neuromuscular junction (NMJ) and corresponding NMJ transmission failure after injury. Here we extend these initial observations at the motor endplate to gain insight into the pre- vs. postsynaptic morphology, as well as the subsynaptic nuclei in healthy (WT) vs. mdx mice. We quantified the discontinuity and branching of the terminal nerve in adult mice. We report mdx- and age-dependent changes for discontinuity and an increase in branching when compared to WT. To examine mdx- and age-dependent changes in the relative localization of pre- and postsynaptic structures, we calculated NMJ occupancy, defined as the ratio of the footprint occupied by presynaptic vesicles vs. that of the underlying motor endplate. The normally congruent coupling between presynaptic and postsynaptic morphology was altered in mdx mice, independent of age. Finally we found an almost two-fold increase in the number of nuclei and an increase in density (nuclei/area) underlying the NMJ. These outcomes suggest substantial remodeling of the NMJ during dystrophic progression. This remodeling reflects plasticity in both pre- and postsynaptic contributors to NMJ structure, and thus perhaps also NM transmission and muscle function. PMID:26441672

  8. Mature mice lacking Rbl2/p130 gene have supernumerary inner ear hair cells and supporting cells.

    PubMed

    Rocha-Sanchez, Sonia M; Scheetz, Laura R; Contreras, Melissa; Weston, Michael D; Korte, Megan; McGee, Joann; Walsh, Edward J

    2011-06-15

    Adult mammalian auditory hair cells (HCs) and their associated supporting cells (SCs) do not proliferate, and HC death leads to irreversible neurosensory hearing loss and balance impairment. In nonmammalian vertebrates, loss of HCs induces mitotic proliferation of adjacent nonsensory SCs and/or direct SC transdifferentiation to generate replacement cells. This results in the structural and functional recovery of the nonmammalian sensory systems. Potential replacement of mammalian auditory HCs, either by transplanting cells or by transforming existing cells through molecular therapy, has long been proposed. However, HC replacement strategies with clear therapeutic potential remain elusive. The retinoblastoma (pRB) family of cell cycle regulators, Rb1, Rbl1 (p107), and Rbl2 (p130), regulate the G(1)- to S-phase transition in proliferating cells. In the inner ear, the biochemical and molecular pathways involving pRBs, particularly p107 and p130, are relatively unexplored and their therapeutic suitability is yet to be determined. In this study, we analyzed the cochleae of adult p130 knock-out (p130(-/-)) mice and showed that lack of the p130 gene results in extra rows of HCs and SCs in the more apical regions of the cochlea. No evidence of transdifferentiation of these supernumerary SCs into HCs was observed in the p130(-/-) mouse. Nevertheless, unscheduled proliferation of SCs in the adult p130(-/-) cochlea coupled to downregulation of bona fide cell cycle inhibitors provides a mechanistic basis for the role of p130 as a regulator of SC and HC mitotic quiescence in the more apical regions of the cochlea. Interestingly, p130(-/-) mice exhibited nearly normal peripheral auditory sensitivity. PMID:21677172

  9. Mature Mice Lacking Rbl2/p130 Gene Have Supernumerary Inner Ear Hair Cells and Supporting Cells

    PubMed Central

    Rocha-Sanchez, Sonia M.; Scheetz, Laura R.; Contreras, Melissa; Weston, Michael D.; Korte, Megan; McGee, JoAnn; Walsh, Edward J.

    2011-01-01

    Adult mammalian auditory hair cells (HCs) and their associated supporting cells (SCs) do not proliferate, and HC death leads to irreversible neurosensory hearing loss and balance impairment. In non-mammalian vertebrates, loss of HCs induces mitotic proliferation of adjacent non-sensory SCs and/or direct SC transdifferentiation to generate replacement cells. This results in the structural and functional recovery of the non-mammalian sensory systems. Potential replacement of mammalian auditory HCs, either by transplanting cells or by transforming existing cells through molecular therapy, has long been proposed. However, HC replacement strategies with clear therapeutic potential remain elusive. The retinoblastoma (pRB) family of cell cycle regulators, Rb1, Rbl1 (p107), and Rbl2 (p130), regulate the G1 to S phase transition in proliferating cells. In the inner ear, the biochemical and molecular pathways involving pRBs, particularly p107 and p130 are relatively unexplored and their therapeutic suitability is yet to be determined. In this study, we analyzed the cochleae of adult p130 knockout (p130−/−) mice and showed that lack of the p130 gene results in extra rows of HCs and SCs in the more apical regions of the cochlea. No evidence of transdifferentiation of these supernumerary SCs into HCs was observed in the p130−/− mouse. Nevertheless, unscheduled proliferation of SCs in the adult p130−/− cochlea coupled to downregulation of bona fide cell cycle inhibitors provides a mechanistic basis for p130’s role as a regulator of SC and HC mitotic quiescence in the more apical regions of the cochlea. Interestingly, p130−/− mice exhibited nearly normal peripheral auditory sensitivity. PMID:21677172

  10. Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse.

    PubMed

    Reay, Daniel P; Niizawa, Gabriela A; Watchko, Jon F; Daood, Molly; Reay, Ja'Nean C; Raggi, Eugene; Clemens, Paula R

    2012-01-01

    Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery. PMID:22231732

  11. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

    PubMed

    Sanderson, N; Factor, V; Nagy, P; Kopp, J; Kondaiah, P; Wakefield, L; Roberts, A B; Sporn, M B; Thorgeirsson, S S

    1995-03-28

    Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine. PMID:7708687

  12. In Vivo Conditional Pax4 Overexpression in Mature Islet β-Cells Prevents Stress-Induced Hyperglycemia in Mice

    PubMed Central

    He, Kai Hui Hu; Lorenzo, Petra I.; Brun, Thierry; Jimenez Moreno, Carmen M.; Aeberhard, Deborah; Ortega, Jorge Vallejo; Cornu, Marion; Thorel, Fabrizio; Gjinovci, Asllan; Thorens, Bernard; Herrera, Pedro L.; Meda, Paolo; Wollheim, Claes B.; Gauthier, Benoit R.

    2011-01-01

    OBJECTIVE To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in β-cells. RESEARCH DESIGN AND METHODS Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression. PMID:21521872

  13. Evaluation of Electrical Impedance as a Biomarker of Myostatin Inhibition in Wild Type and Muscular Dystrophy Mice

    PubMed Central

    Sanchez, Benjamin; Li, Jia; Yim, Sung; Pacheck, Adam; Widrick, Jeffrey J.; Rutkove, Seward B.

    2015-01-01

    Objectives Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx). Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters. Methods Eight wild-type (wt) and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg−1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed. Results As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p < 0.001 respectively) and muscle mass (25% p < 0.05 and 22% p < 0.001, respectively). The Cole impedance parameters in treated wt mice, showed a 24% lower central frequency (p < 0.05) and 19% higher resistance ratio (p < 0.05); no significant differences were observed in the mdx mice. These differences were consistent with those seen in maximum isometric force, which was greater in the wt animals (p < 0.05 at > 70 Hz), but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01). Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively). Conclusions Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials. PMID:26485280

  14. Assessing functional performance in the mdx mouse model.

    PubMed

    Aartsma-Rus, Annemieke; van Putten, Maaike

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories. PMID:24747372

  15. Assessing Functional Performance in the Mdx Mouse Model

    PubMed Central

    Aartsma-Rus, Annemieke; van Putten, Maaike

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories. PMID:24747372

  16. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

    PubMed Central

    Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang

    2016-01-01

    Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods. PMID:26966678

  17. Dystropathology increases energy expenditure and protein turnover in the Mdx mouse model of Duchenne muscular dystrophy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the diet...

  18. Sensitive ultrasonic delineation of steroid treatment in living dystrophic mice with energy-based and entropy-based radio frequency signal processing.

    PubMed

    Wallace, Kirk D; Marsh, Jon N; Baldwin, Steven L; Connolly, Anne M; Keeling, Richard; Lanza, Gregory M; Wickline, Samuel A; Hughes, Michael S

    2007-11-01

    Duchenne muscular dystrophy is a severe wasting disease, involving replacement of necrotic muscle tissue by fibrous material and fatty infiltrates. One primary animal model of this human disease is the X chromosome-linked mdx strain of mice. The goals of the present work were to validate and quantify the capability of both energy and entropy metrics of radio-frequency ultrasonic backscatter to differentiate among normal, dystrophic, and steroid-treated skeletal muscle in the mdx model. Thirteen 12-month-old mice were blocked into three groups: 4 treated mdx-dystrophic that received daily subcutaneous steroid (prednisolone) treatment for 14 days, 4 positive-control mdx-dystrophic that received saline injections for 14 days, and 5 negative-control animals. Biceps muscle of each animal was imaged in vivo using a 40-MHz center frequency transducer in conjunction with a Vevo-660 ultrasound system. Radio-frequency data were acquired (1 GHz, 8 bits) corresponding to a sequence of transverse images, advancing the transducer from "shoulder" to "elbow" in 100-micron steps. Data were processed to generate both "integrated backscatter" (log energy), and "entropy" (information theoretic receiver, H(f)) representations. Analyses of the integrated-backscatter values delineated both treated-and untreated-mdx biceps from normal controls (p < 0.01). Complementary analyses of the entropy images differentiated the steroid-treated and positive-control mdx groups (p < 0.01). To our knowledge, this study represents the first reported use of quantitative ultrasonic characterization of skeletal muscle in mdx mice. Successful differentiation among dystrophic, steroid-treated, and normal tissues suggests the potential for local noninvasive monitoring of disease severity and therapeutic effects. PMID:18051163

  19. Differentiation of murine embryonic stem cells in skeletal muscles of mice.

    PubMed

    Tian, Chai; Lu, Yifan; Gilbert, Rénald; Karpati, George

    2008-01-01

    Possible myogenic differentiation of SSEA-1- and OCT-4-positive murine embryonic stem cells (ESCs) and embryoid bodies (EBs) was studied in vitro and in vivo. In vitro, ESC- or EB-derived ESCs (EBs/ESCs) showed only traces of Pax 3 and 7 expression by immunocytochemistry and Pax 3 expression by immunoblot. By RT-PCR, myogenic determinant molecules (myf5, myoD, and myogenin) were expressed by EBs/ESCs but not by ESCs. However, in such cultures, very rare contracting myotubes were still present. Suspensions of LacZ-labeled ESCs or EBs were injected into anterior tibialis muscles (ATM) of different cohorts of mice for the study of their survival and possible myogenic differentiation. The different cohorts of mice included isogenic adult 129/Sv, nonisogenic CD1 and mdx, as well as mdx immunosuppressed with 2.5 mg/kg daily injections of tacrolimus. Ten to 90 days postinjections, the injected ATM of nonisogenic mice did not contain cells positive for LacZ, SSEA-1, OCT-4, or embryonic myosin heavy chain. The ATM of intact mdx mice contained very rare examples of muscle fibers positive for dystrophin and/or embryonic myosin heavy chain. In the ATM of the isogenic normal and the immunosuppressed mdx mice, as expected, large teratomas developed containing the usual diverse cell types. In some teratomas of immunosuppressed mdx mice, small pockets of muscle fibers expressed dystrophin and myosin heavy chain. Our studies indicated that in muscles of animals nonisogenic with the used ESCs, only very rare ESCs survived with myogenic differentiation. These studies also indicated that ESCs will not undergo significant, selective, and preferential myogenic differentiation in vitro or in vivo in any of the models studied. It is probable that this strain of murine ESC requires some experimentally induced alteration of its gene expression profile to secure significant myogenicity and suppress tumorogenicity. PMID:18522235

  20. Systemic Trans-splicing adeno-associated viral delivery efficiently transduces the heart of adult mdx mouse, a model for duchenne muscular dystrophy.

    PubMed

    Ghosh, Arkasubhra; Yue, Yongping; Shin, Jin-Hong; Duan, Dongsheng

    2009-11-01

    Trans-splicing adeno-associated viral (tsAAV) vectors hold great promise for delivering large therapeutic genes. One potential application is in the treatment of Duchenne muscular dystrophy (DMD). In this case, it is necessary to transduce whole body muscle. We demonstrated body-wide AAV-9 tsAAV transduction in normal neonatal mice. However, it was not clear whether such an approach would work in diseased mice. In this study we delivered the AAV-9 alkaline phosphatase (AP) tsAAV vector (3 x 10(12) vector genome particles per vector per mouse, tail vein injection) to 2-month-old mdx mice, the most widely used DMD model. Four months later, we observed widespread AP expression in the heart. It reached the same level as we have seen in normal neonatal puppy. Interestingly, myocardial transduction correlated with beta-myosin heavy chain expression but not with LamR, the putative AAV-9 receptor. AP expression was also detected in various skeletal muscles but at levels much lower than in normal newborn mice. Despite the existing inflammatory milieu, we did not see any appreciable increase in CD4(+) and CD8(+) T cells and macrophages in striated muscles after systemic tsAAV infection. In summary, our results have paved the way for tsAAV-mediated gene therapy for Duchenne cardiomyopathy. PMID:19627234

  1. Minor influence of lifelong voluntary exercise on composition, structure, and incidence of osteoarthritis in tibial articular cartilage of mice compared with major effects caused by growth, maturation, and aging.

    PubMed

    Närhi, Tommi; Siitonen, Ulrika; Lehto, Lauri J; Hyttinen, Mika M; Arokoski, Jari P A; Brama, Pieter A; Jurvelin, Jukka S; Helminen, Heikki J; Julkunen, Petro

    2011-10-01

    We investigated the effects of lifelong voluntary exercise on articular cartilage of mice. At the age of 4 weeks C57BL mice (n = 152) were divided into two groups, with one group serving as a sedentary control whereas the other was allowed free access to a running wheel from the age of 1 month onward. Mice were euthanized at four different time points (1, 2, 6, and 18 months of age). Articular cartilage samples were gathered from the load-bearing area of the tibial medial plateaus, and osteoarthritis was graded. Additionally, the proteoglycan content distribution was assessed using digital densitometry, collagen fibril orientation, and parallelism with polarized light microscopy, and collagen content using Fourier transform infrared imaging spectroscopy. The incidence of osteoarthritis increased with aging, but exercise had no effect on this trend. Furthermore, the structure and composition revealed significant growth, maturation, and age-dependent properties. Exercise exerted a minor effect on collagen fibril orientation in the superficial zone. Fibril orientation at 2 months of age was more perpendicular to surface (p < 0.05) in controls compared with runners, whereas the situation was reversed at the age of 18 months (p < 0.05). The collagen content of the superficial zone was higher (p < 0.01) at the age of 18 months in controls compared with runners but the proteoglycan content did not display any exercise-dependent changes. In conclusion, growth, maturation, and aging exerted a clear effect on integrity, structure, and composition of medial tibial plateau articular cartilage in mice, whereas lifelong voluntary exercise had only a minor effect on collagen architecture and content. PMID:21405978

  2. Absence of intracellular ion channels TPC1 and TPC2 leads to mature-onset obesity in male mice, due to impaired lipid availability for thermogenesis in brown adipose tissue.

    PubMed

    Lear, Pamela V; González-Touceda, David; Porteiro Couto, Begoña; Viaño, Patricia; Guymer, Vanessa; Remzova, Elena; Tunn, Ruth; Chalasani, Annapurna; García-Caballero, Tomás; Hargreaves, Iain P; Tynan, Patricia W; Christian, Helen C; Nogueiras, Rubén; Parrington, John; Diéguez, Carlos

    2015-03-01

    Intracellular calcium-permeable channels have been implicated in thermogenic function of murine brown and brite/beige adipocytes, respectively transient receptor potential melastin-8 and transient receptor potential vanilloid-4. Because the endo-lysosomal two-pore channels (TPCs) have also been ascribed with metabolic functionality, we studied the effect of simultaneously knocking out TPC1 and TPC2 on body composition and energy balance in male mice fed a chow diet. Compared with wild-type mice, TPC1 and TPC2 double knockout (Tpcn1/2(-/-)) animals had a higher respiratory quotient and became obese between 6 and 9 months of age. Although food intake was unaltered, interscapular brown adipose tissue (BAT) maximal temperature and lean-mass adjusted oxygen consumption were lower in Tpcn1/2(-/-) than in wild type mice. Phosphorylated hormone-sensitive lipase expression, lipid density and expression of β-adrenergic receptors were also lower in Tpcn1/2(-/-) BAT, whereas mitochondrial respiratory chain function and uncoupling protein-1 expression remained intact. We conclude that Tpcn1/2(-/-) mice show mature-onset obesity due to reduced lipid availability and use, and a defect in β-adrenergic receptor signaling, leading to impaired thermogenic activity, in BAT. PMID:25545384

  3. Utrophins compensate for Dp71 absence in mdx3cv in adhered platelets.

    PubMed

    Cerecedo, Doris; Mondragón, Ricardo; Candelario, Aurora; García-Sierra, Francisco; Mornet, Dominique; Rendón, Alvaro; Martínez-Rojas, Dalila

    2008-01-01

    Platelet adhesion is a critical step due to its hemostatic role in stopping bleeding after vascular damage. Short dystrophins are the most abundant dmd gene products in nonmuscle tissues, and in association with cytoskeleton proteins contribute to their intrinsic function; while utrophins are dystrophin-homologous related family proteins with structural and functional similarities. We previously demonstrated the presence of Dp71 isoforms, utrophins, and various dystrophin-associated proteins and their participation in cytoskeleton re-organization, filopodia and lamellipodia extension, and in centralizing cytoplasmic granules during the adhesion process of human platelets. To evaluate the morphologic changes and actin-based structures of mdx(3cv) platelets during the adhesion process, we compared the topographic distribution of Dp71d/Dp71Delta110(m) and dystrophin-associated protein in adhered platelets from dystrophic mdx(3cv) mouse. By confocal microscopy, we showed that absence of Dp71 isoforms in platelets from this animal model disrupted dystrophin-associated protein expression and distribution without modifying the platelet morphology displayed during the glass-adhesion process. By immunoprecipitation assays, we proved that up-regulated utrophins were associated with dystrophin-associated proteins to conform the dystrophin-associated protein complex corresponding to utrophins, which might compensate for Dp71 absence in mdx(3cv) platelets. PMID:18180614

  4. The mdx Mutation in the 129/Sv Background Results in a Milder Phenotype: Transcriptome Comparative Analysis Searching for the Protective Factors

    PubMed Central

    Calyjur, Priscila Clara; Almeida, Camila de Freitas; Ayub-Guerrieri, Danielle; Ribeiro, Antonio Fernando; Fernandes, Stephanie de Alcântara; Ishiba, Renata; dos Santos, Andre Luis Fernandes; Onofre-Oliveira, Paula; Vainzof, Mariz

    2016-01-01

    The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process. PMID:26954670

  5. A PCR-based assay for the wild-type dystrophin gene transferred into the mdx mouse.

    PubMed

    Shrager, J B; Naji, A; Kelly, A M; Stedman, H H

    1992-10-01

    Myoblast transfer has emerged as a promising treatment for inherited myopathies such as Duchenne muscular dystrophy (DMD). Further development of the technique's therapeutic potential requires an experimental system in which issues of graft rejection can be clearly discriminated from those related to myoblast biology. Here we report the development and initial application of a quantitative assay for myogenic cells bearing a wild-type dystrophin gene following transfer into the mdx mouse. The technique relies upon the ability of a mutagenizing polymerase chain reaction (PCR) primer to create a new restriction site in the amplification production of the wild-type, but not the mdx dystrophin gene. The ratio of host to donor cells can be determined from muscle biopsies as small as 1 mg, regardless of donor H-2 background. This simple technique should allow a number of basic questions related to myoblast and direct gene transfer to be addressed using the mdx mouse model. PMID:1357549

  6. Sox2-CreER mice are useful for fate mapping of mature, but not neonatal, cochlear supporting cells in hair cell regeneration studies

    PubMed Central

    Walters, Bradley J.; Yamashita, Tetsuji; Zuo, Jian

    2015-01-01

    Studies of hair cell regeneration in the postnatal cochlea rely on fate mapping of supporting cells. Here we characterized a Sox2-CreER knock-in mouse line with two independent reporter mouse strains at neonatal and mature ages. Regardless of induction age, reporter expression was robust, with CreER activity being readily detectable in >85% of supporting cells within the organ of Corti. When induced at postnatal day (P) 28, Sox2-CreER activity was exclusive to supporting cells demonstrating its utility for fate mapping studies beyond this age. However, when induced at P1, Sox2-CreER activity was also detected in >50% of cochlear hair cells, suggesting that Sox2-CreER may not be useful to fate map a supporting cell origin of regenerated hair cells if induced at neonatal ages. Given that this model is currently in use by several investigators for fate mapping purposes, and may be adopted by others in the future, our finding that current protocols are effective for restricting CreER activity to supporting cells at mature but not neonatal ages is both significant and timely. PMID:26108463

  7. Market maturity

    SciTech Connect

    Meade, B.; Bowden, S.; Ellis, M

    1995-02-01

    The power sector in the Philipines provides one of the most mature independent power markets in Asia. Over the past five years, National Power Corp. (NPC), the government owned utility, has actively invited the power sector into power generation. Distribution has remained in the hands of private and rural cooperative utilities. Private utilities have been operating as full requirements customers of NPC while the growth in capacity additions by independent power producers (IPPs) has outpaced NPC`s for the second year in a row. With a recovering economy and regulatory reform proceeding, the outlook for independent power remains strong through the end of the decade. The Philipine Congress is now reviewing draft legislation that will decentralize NPC and begin the process of privatization and market-based reforms throughout the country`s power sector.

  8. Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

    PubMed

    Zhao, Chunli; Farruggio, Alfonso P; Bjornson, Christopher R R; Chavez, Christopher L; Geisinger, Jonathan M; Neal, Tawny L; Karow, Marisa; Calos, Michele P

    2014-01-01

    A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies. PMID:24781921

  9. Mechanisms inducing low bone density in Duchenne muscular dystrophy in mice and humans.

    PubMed

    Rufo, Anna; Del Fattore, Andrea; Capulli, Mattia; Carvello, Francesco; De Pasquale, Loredana; Ferrari, Serge; Pierroz, Dominique; Morandi, Lucia; De Simone, Michele; Rucci, Nadia; Bertini, Enrico; Bianchi, Maria Luisa; De Benedetti, Fabrizio; Teti, Anna

    2011-08-01

    Patients affected by Duchenne muscular dystrophy (DMD) and dystrophic MDX mice were investigated in this study for their bone phenotype and systemic regulators of bone turnover. Micro-computed tomographic (µCT) and histomorphometric analyses showed reduced bone mass and higher osteoclast and bone resorption parameters in MDX mice compared with wild-type mice, whereas osteoblast parameters and mineral apposition rate were lower. In a panel of circulating pro-osteoclastogenic cytokines evaluated in the MDX sera, interleukin 6 (IL-6) was increased compared with wild-type mice. Likewise, DMD patients showed low bone mineral density (BMD) Z-scores and high bone-resorption marker and serum IL-6. Human primary osteoblasts from healthy donors incubated with 10% sera from DMD patients showed decreased nodule mineralization. Many osteogenic genes were downregulated in these cultures, including osterix and osteocalcin, by a mechanism blunted by an IL-6-neutralizing antibody. In contrast, the mRNAs of osteoclastogenic cytokines IL6, IL11, inhibin-βA, and TGFβ2 were increased, although only IL-6 was found to be high in the circulation. Consistently, enhancement of osteoclastogenesis was noted in cultures of circulating mononuclear precursors from DMD patients or from healthy donors cultured in the presence of DMD sera or IL-6. Circulating IL-6 also played a dominant role in osteoclast formation because ex vivo wild-type calvarial bones cultured with 10% sera of MDX mice showed increase osteoclast and bone-resorption parameters that were dampen by treatment with an IL-6 antibody. These results point to IL-6 as an important mediator of bone loss in DMD and suggest that targeted anti-IL-6 therapy may have a positive impact on the bone phenotype in these patients. PMID:21509823

  10. Toward Integrated Molecular Diagnostic System (iMDx): Principles and Applications

    PubMed Central

    Park, Seung-min; Sabour, Andrew F.; Son, Jun Ho; Lee, Sang Hun

    2014-01-01

    Integrated molecular diagnostic systems (iMDx), which are automated, sensitive, specific, user-friendly, robust, rapid, easy-to-use, and portable, can revolutionize future medicine. This review will first focus on the components of sample extraction, preservation, and filtration necessary for all point-of-care devices to include for practical use. Subsequently, we will look for low-powered and precise methods for both sample amplification and signal transduction, going in-depth to the details behind their principles. The final field of total device integration and its application to the clinical field will also be addressed to discuss the practicality for future patient care. We envision that microfluidic systems hold the potential to breakthrough the number of problems brought into the field of medical diagnosis today. PMID:24759281