Short-term treatment with Uncaria tomentosa aggravates the injury phenotype in mdx mice

Introduction: Uncaria tomentosa (Willd. ex Roem. & Schult.) DC. (Rubiaceae) or UT is a medicinal plant with antiviral, antimutagenic, anti-inflammatory and antioxidant properties. Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene; this deficiency leads to sarcolemma instability, inflammation, muscle degeneration and fibrosis. Objective: Considering the importance of inflammation to dystrophy progression and the anti-inflammatory activity of UT, in the present study we evaluated whether oral administration of UT extract would ameliorate dystrophy in the mdx mice, a DMD model. Methods: Eight-week-old male mdx mice were submitted to 200 mg/kg body weight daily UT oral administration for 6 weeks. General histopathology was analysed, and muscle tumor necrosis factor α, transforming growth factor-ß, myostatin and osteopontin transcript levels were assessed. The ability of mice to sustain limb tension to oppose their gravitational force was measured. Data were analysed with the unpaired Student's t-test. Results: Morphologically, both untreated and UT-treated animals exhibited internalised nuclei, increased endomysial connective tissue and variations in muscle fibre diameters. Body weight and muscle strength were significantly reduced in the UT-treated animals. Blood creatine kinase was higher in UT-treated compared to untreated animals. In tibialis anterior, myostatin, transcript was more highly expressed in the UT-treated while in the diaphragm muscle, transforming growth factor-ß transcripts were less expressed in the UT-treated. Conclusion: While previous studies identified anti-inflammatory, antiproliferative and anticarcinogenic UT effects, the extract indicates worsening of dystrophic muscles phenotype after short-term treatment in mdx mice.

Lack of dystrophin influences muscle inflammation but not myogenic regulatory factors after eccentric exercise in mdx mice

Abstract Aim: To investigate the consequences of chronic eccentric exercise in histopathology, inflammatory, and myogenic regulatory factors response in gastrocnemius muscle of X-chromosome-linked muscular dystrophy (mdx) mice. Method: Male mdx and control mice (C57BL/10 lineage) were distributed in the following groups: Sedentary Control (SC), Trained Control (TC), Sedentary Mdx (S-Mdx), and Trained Mdx (T-Mdx). Trained animals were subjected to downhill running for 7 weeks. Gastrocnemius was submitted to histopathological analysis and immunoexpression of Cyclooxygenase-2 (COX-2) and myogenic regulatory factors (myoD and myogenin). Results: The exercise influenced inflammation response as demonstrated by the increased COX-2 immunoexpression in T-Mdx. Interestingly, Myogenic regulatory factors revealed that the lack of dystrophin has not been influenced myoD and the increase of myogenin occurred due to exercise and was not aggravated by the absence of dystrophin. Conclusion: In conclusion, an eccentric exercise in gastrocnemius of mdx mice was characterized by an intense inflammatory process without myogenic response. These findings suggest that special attention should be given to inflammatory aspects related to COX-2 associated with a decrease of myoD expression, as biomarkers in motor rehabilitation programs.

Arquitetura comparativa dos pulmões de camundongos normais e afetados pela Distrofia Muscular de Duchenne / Comparative lung architecture of normal and by Duchenne Muscular Dystrophy affected mice

A Distrofia Muscular de Duchenne (DMD) é uma doença genética de caráter recessivo que caracterizada por fraqueza muscular progressiva de cintura pélvica e escapular evoluindo para insuficiência respiratória e, ou cardíaca. O camundongo mdx é um modelo amplamente utilizado para estudos da DMD. Apesar do fenótipo destes animais serem mais suave, estes apresentam o principal músculo respiratório, o diafragma com morfologia e bioquímica semelhante à DMD humana, fato este que pode comprometer a função respiratória e consequentemente os pulmões. Foi realizado um estudo anatômico descritivo do parênquima pulmonar dos pulmões de 5 animais modelo mdx comparando estes com os pulmões de 5 camundongos BALB/C57 (Mus musculus). Os pulmões foram analisados macroscopicamente e através de microscopia de luz e eletrônica de varredura. Os achados sugerem que o modelo mdx apresenta morfologia pulmonar semelhante aos camundongos BALB/C57 e que seu uso deve ser cauteloso e criterioso em ensaios clínicos que aborde este órgão.(AU)

The Duchenne Muscular Dystrophy (DMD) is a recessive genetic disease characterized by progressive muscle weakness of the pelvic and scapular girdle and progressing to respiratory or heart failure. The mdx mouse is a model widely used for studies. Although they possess a milder phenotype, the morphology and biochemistry of the diaphragm are similar to human DMD. We performed a descriptive anatomical study of the pulmonary parenchyma of five mdx animal models and compared these with the lungs of 5 mice BALB/C57 (Mus musculus). The findings suggest that the mdx model has morphological features similar to BALB/C57 mice and it must be used with caution in clinical trials which involve the lung.(AU)

Arquitetura comparativa dos pulmões de camundongos normais e afetados pela Distrofia Muscular de Duchenne

Abstract: The Duchenne Muscular Dystrophy (DMD) is a recessive genetic disease characterized by progressive muscle weakness of the pelvic and scapular girdle and progressing to respiratory or heart failure. The mdx mouse is a model widely used for studies. Although they possess a milder phenotype, the morphology and biochemistry of the diaphragm are similar to human DMD. We performed a descriptive anatomical study of the pulmonary parenchyma of five mdx animal models and compared these with the lungs of 5 mice BALB/C57 (Mus musculus). The findings suggest that the mdx model has morphological features similar to BALB/C57 mice and it must be used with caution in clinical trials which involve the lung.

Resumo: A Distrofia Muscular de Duchenne (DMD) é uma doença genética de caráter recessivo que caracterizada por fraqueza muscular progressiva de cintura pélvica e escapular evoluindo para insuficiência respiratória e, ou cardíaca. O camundongo mdx é um modelo amplamente utilizado para estudos da DMD. Apesar do fenótipo destes animais serem mais suave, estes apresentam o principal músculo respiratório, o diafragma com morfologia e bioquímica semelhante à DMD humana, fato este que pode comprometer a função respiratória e consequentemente os pulmões. Foi realizado um estudo anatômico descritivo do parênquima pulmonar dos pulmões de 5 animais modelo mdx comparando estes com os pulmões de 5 camundongos BALB/C57 (Mus musculus). Os pulmões foram analisados macroscopicamente e através de microscopia de luz e eletrônica de varredura. Os achados sugerem que o modelo mdx apresenta morfologia pulmonar semelhante aos camundongos BALB/C57 e que seu uso deve ser cauteloso e criterioso em ensaios clínicos que aborde este órgão.

Effect of Citrus aurantium L. essential oil on muscle regeneration in mdx mice / Efecto del aceite esencial de Citrus aurantium L. en la regeneración muscular en ratones mdx

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder characterized by the progressive loss of muscular strength. Mdx mutant mice show a marked deficiency in dystrophin, which was related to muscle membrane stability. The aim of this study was to verify the possible protective anti-inflammatory effect of citrus oil on mdx muscle fibers. Thus, adult male and female mdx mice (014/06-CEEA) were divided into control and citrus-treated. After 60 days of treatment, one ml of blood was collected for creatine kinase (CK) test. Diaphragm, sternomastoideus, anterior tibial and gastrocnemius muscles were removed and processed according to histological routine methods. The observed alterations indicate a direct effect of citrus. Recent studies have improved the diagnosis of muscular diseases but with no definitions of efficient treatments. Intervention with several therapies is important to many patients presenting muscular dystrophy, which enables them to live longer and be more active, while there is no development of gene therapies.

La distrofia muscular de Duchenne (DMD) es una enfermedad grave ligada al cromosoma X, trastorno recesivo que se caracteriza por la pérdida progresiva de fuerza muscular. Mdx ratones mutantes muestran una marcada deficiencia en la distrofina, que está relacionada con la estabilidad de la membrana muscular. El objetivo de este estudio fue comprobar el posible efecto protector, antiinflamatorio del aceite de cítricos en las fibras musculares mdx. Los ratones mdx adultos machos y hembras (014/06-CEEA) se dividieron en control y cítricos tratados. Después de 60 días de tratamiento, un ml de sangre fue recogida para cuantificar la creatina quinasa (CK) de prueba. Fueron retirados y procesados los músculos diafragma, esternomastoideo, tibial anterior y gastrocnemio de acuerdo con los métodos de rutina histológica. Las alteraciones observadas indican un efecto directo de los cítricos. Estudios recientes han mejorado el diagnóstico de enfermedades musculares, pero sin definiciones de tratamientos eficaces. Intervención con varias terapias es importante para muchos pacientes que presentan distrofia muscular, lo que les permite vivir más y ser más activos, mientras no exista desarrollo de terapias génicas.

Qualitative analysis of striated skeletal muscles in thedystrophynopathy of mdx mice submitted to physical activity

Duchenne’s Muscular Dystrophy (DMD) is a recessive hereditary myopathy linked to the chromosome X,caused by a mutation in the dystrophin gene, which strengthens and stabilizes the sarcolemma during the stressof muscular contraction and, when absent, the sarcollema ruptures and allows calcium to enter, which causesthe muscle fiber to necrotize. The object of the present study was to perform the morphologic analysis of theanterior tibial and the gastrocnemius muscles with (w/pa) or without (n/pa) physical activity for five weeks.We used 72 mice, divided in 12 experimental groups – 6 of them mdx and 6 control groups (C57/10J) aged4, 7, and 10 weeks. The samples were collected, processed and stained with hematoxylin-eosin. They wereanalyzed by light microscopy, selected and photomicrographed. On the cross-sections of control animals aged4, 7 and 10 weeks w/pa and n/pa, polygonal muscle fibers of many different sizes, ellipse-shaped and withseveral peripheral nucleuses were observed. In the mdx mice w/pa aged 4, 7 and 10 weeks, the muscle fibersshowed different shapes, sizes and stain affinities, rounded edges, anuclear or centered nucleuses; hyalines andmyofibrillas were highly contracted. Muscular regeneration and nectrotic areas with inflammatory infiltrateswere identified in the mdx animals aged 4, 7 and 10 weeks w/pa, as well as in animals aged 10 weeks n/pa.With the progression of the disease in the animals submitted to physical activity, there was evidence of failurein the regeneration and muscular degeneration, intensified and characterized by the gradual replacement ofthe striated skeletal muscle tissue by fibroadipose connective tissue.

The Effect of Steroid on Heat Shock Protein 70 Expression in mdx Mice

OBJECTIVE: To investigate the effect of steroid administration on the apoptosis and heat shock protein 70 (HSP70) expression after exercise in the animal model of Duchenne muscular dystrophy. METHOD: We measured Bcl-2, BAX and HSP70 expression by western blotting. 20 control and 20 mdx mice were divided into free-living (n=10) and exercise (n=10) groups. Free-living and exercise groups were further divided into steroid-treated and sham-treated groups to evaluate the effect of steroid administration. RESULTS: Apoptosis was most prominent in the sham-treated exercise group, while apoptosis was significantly reduced in the steroid-treated exercise group. HSP70 expression was maximized in sham-treated exercise group, whereas steroid administration inhibited HSP70 expression after exercise in muscular dystrophy animal model. Exercise loading was found to cause severe apoptosis but steroid administration alleviated apoptotic damage in mdx mice. CONCLUSION: HSP70 expression was suppressed in the steroid-treated exercise group, which suggests steroid might have major preventive effect in exercise-induced apoptosis of muscular dystrophy animal model.

The Effect of L-Carnitine Supplementation on the Dystrophic Muscle and Exercise Tolerance of Muscular Dystrophy (mdx) Mice

BACKGROUND: Duchenne muscular dystrophy is an X-linked recessive disorder leading to death in the late teens or early twenties. There is no effective pharmacological therapy for now. L-carnitine (LCAR), a naturally occurring compound facilitating the transport of fatty acid into mitochondria for -oxidation, has been getting an attention for its antiapoptotic and osmoprotective effect. The aim of this study is to evaluate if LCAR administration reduces dystrophic progression and enhances exercise tolerance in dystrophin deficient (mdx) mice. METHODS: Mdx mice (n=5) and wild type mice (n=5), aged 3 weeks were treated with oral LCAR (75mg/kg/day) for 6 weeks. Five each mdx and wild type mice were recruited for their counter-control. The animals underwent a 30-minute run on a horizontal treadmill for evaluating their exercise endurance. After 6-week training, baseline and post exercise serum CK of each group were analyzed. We examined sarcolemma integrity and muscle histology after exercise. Immunofluorescent stain and Western blot analysis for dystrophin-dystroglycan complex were also performed. RESULTS: LCAR-treated mdx mice showed higher exercise tolerance and lower serum CK value compared with those of control mice. The area of Evans blue dye uptake in LCAR-treated mdx mice was much smaller than that of control mdx mice. There was no remarkable difference in dystrophin-dystroglycan complex expression between treated and control mdx mice. CONCLUSIONS: LCAR seems to enhance exercise tolerance and decrease the breakdown of sarcolemma during strenuous exercise. Our study suggests the possibility of adjunctive therapeutic use of L-carnitine to the patients with Duchenne muscular dystrophy.

Effects of Exercise and Steroid on Apoptosis in Skeletal Muscle of Mdx Mouse

OBJECTIVE: To investigate the effect of exercise and steroid to the muscle of animal model of Duchenne muscular dystrophy. METHOD: We used 15 mdx and 15 control mice. To grade exercise loading, control and mdx mice were divided into free-living, exercise and immobilization groups. Free-living and exercise groups were further divided into steroid-treated and sham-treated groups to evaluate the effect of steroid administration. We measured the apoptotic changes using in situ DNA nick-end labling (TUNEL), DNA fragmentation assay and western blots for Bcl-2 and BAX. RESULT: With TUNEL method, the largest number of myonuclei became positive in sham-treated exercise group while apoptosis was significantly reduced in steroid-treated exercise group in mdx mice. Steroid-treated free-living group showed higher rate of apoptotic change than sham-treated free-living group. With western blots for Bcl-2 and BAX, the value of BAX/Bcl-2 ratio was highest in sham-treated exercise group and among free living mdx mice, it was higher in steroid-treated group than sham-treated one. CONCLUSION: Apoptosis can be minimized in free living condition while exercise loading or immobilization can cause apoptotic change in muscular dystrophy animal model. Steroid administration induces apoptosis in free living muscle and it alleviates apoptotic damage caused by exercise loading in mdx mice.

Myoblast Transfer Therapy on mdxMouse / 대한정형외과학회잡지

PURPOSE: To observe dystrophin formation and histological improvement in dystrophic muscle of mdx mouse after normal myoblast injection. MATERIALS AND METHODS: Cultured myoblasts from genetically normal rats were injected into the right quadriceps femoris of a 6-week-old mdx mouse (n=9). dPBS was injected into the left quadriceps femoris as a control. One, 2, and 3 months after injection, The control and experimental group were compared histologically and by dystrophin immunostaining. RESULTS: When compared with controls 3 months postoperatively, quadriceps femoris in the experimental group exhibited greater cross-sectional area and total fiber number, and the experimental animals contained more normal-appearing and less abnormalappearing fibers than the control group. Most of the fibers in the experimental group showed positive results in dystrophin immunostaining, whereas immunostaining of mdx muscle fibers in the control group was completely negative. CONCLUSION: This study shows that normal myoblast injection improved the muscle architecture histologically and produced dystrophin protein in dystrophic muscle.

Inactivation of mammalian X-chromosome during spermatogenesis: Temporal expression of genes in the laboratory mouse.

At zygotene/pachytene stage of meiosis in mammalian testis, the X—Y heterobivalent is sequesterd into a heterochromatinized body whose genetic inactivity is shown by lack of uridine incorporation. For the genic level evaluation of the X-inactivation, activities of three X-linked genes were assayed in testicular cell types in the laboratory mouse. While hypoxanthine phosphoribosyl transferase is functional at least up to pachytene stage of primary spermatocytes, glucose-6-phosphate dehydrogenase appears to be active only in the Sertoli cells. No transcript of the muscle specific muscular dystrophin gene was obtained from its regular 5' promoter. Thus, inactivation of X-linked genes in testis occurs at different stages during spermatogenesis, independently of heterochrornatinizati on of the XY-body. We propose that Xist transcript, the putative regulator of X-inactivation in female soma, is also the regulator in testis. However, due to its extremely low level in testis the transcript may regulate by "spreading" in a gradient and affect the genes in a temporal order. Thus, besides other factors, physical proximity of the genes to Xist may determine the stage of their inactivation.