Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease affecting 1 in every 3500 boys. The disease is characterized by the absence of the dystrophin protein from the sarcolemma of muscle cells. Muscle cells lacking dystrophin go through cycles of degeneration and regeneration and are considered susceptible to contraction-induced injury 144. Eventually, the satellite cell proliferative capacity is exhausted and the muscle fibers are replaced by connective and adipose tissue that yields a progressive loss of force generating capability. DMD patients typically die by their early 20's, primarily due to respiratory or cardiac failure. The precise role of dystrophin is not presently known. However, its absence suggests that it may play a role in both cellular calcium regulation and oxidative stress 152. Recent studies suggest increased reactive oxygen species (ROS) may precede the initial wave of wasting that marks disease onset 49. Therefore, it is possible oxidative stress may contribute as a pathogenic mechanism of DMD. Strategies to reduce the deleterious effects of oxidative stress could be an effective therapeutic approach. Regular exercise is known to increase antioxidant capacity in humans and mice 146. Green tea extract (GTE) is a powerful antioxidant that is easily supplemented in the diet 83. The purpose of this study was to test the hypotheses that (1) voluntary endurance exercise alone, (2) a diet supplemented with 0.05% (wt/wt) GTE alone, or exercise and GTE combined will blunt the effects of ROS and improve muscle strength and endurance in young mdx mice. Male mdx mice at age 21-days were randomly divided into one of 4 treatment groups: Run Normal diet (RunNorm; n=8), Sedentary Normal diet (SedNorm; n=8), Run GTE diet (RunGTE; n=10), and Sedentary GTE diet (SedGTE; n=8). RunNorm and RunGTE mice were given free access to a running wheel for 3 weeks while SedNorm and SedGTE mice were restricted to normal cage movement. At the end of 3 weeks, mice in each treatment group were sacrificed and assessed for daily and weekly running distances, content of actin and myosin proteins and fiber type distribution (tibialis anterior), contractile/mechanical and fatigue properties (extensor digitorum longus), creatine kinase levels and antioxidant capacity (serum), lipid peroxidation (gastrocnemius), and citrate synthase and beta-hydroxyacyl-CoA dehydrogenase activities (quadriceps and soleus). The key findings of this study were: In normal diet running mice (RunNorm), average daily distance run was increased 300% (from 0.5 to 2.1 km/d, P<0.05) from week 1 to week 3. In GTE diet (RunGTE) compared to RunNorm mice, total distance over the 3 weeks was markedly improved 128% (61.2 vs. 26.8 km, P<0.0001). Running, independent of diet increased EDL muscle tetanic stress (18%), serum antioxidant capacity (22%), citrate synthase activity (35%), and beta-oxidation (37%; all P<0.05). GTE, independent of running decreased lipid peroxidation (gastrocnemius:-64%; heart: -29%) and serum creatine kinase (-36%), and increased citrate synthase activity (59%; all P<0.05). These findings in dystrophic mice suggest that voluntary endurance exercise with or without GTE supplementation blunted the deleterious effects of ROS. If similar positive effects are evident in human DMD patients, then these approaches may be beneficial therapies either alone or in combination. / Master of Science

Essentielle à l'équilibre énergétique de la cellule, la mitochondrie, véritable sentinelle, joue, un rôle majeur dans le destin de la cellule, en modulant les voies de signalisation de mort cellulaire mis en jeu dans l'atrophie musculaire. L'objectif de cette thèse est de proposer des cibles thérapeutiques centrées sur la mitochondrie dans deux modèles murins dont la physiopathologie est caractérisée par une dysfonction mitochondriale associée à une atrophie musculaire : le vieillissement et la dystrophie musculaire de Duchenne (DMD). Pour lutter contre la perte de masse musculaire liée à l'âge, la déficience en myostatine (mstn), associée à un phénotype hypermusculé, est une stratégie thérapeutique prometteuse. Mais, l'altération du métabolisme mitochondrial et oxydatif induite par cette déficience réduit les effets bénéfiques d'une telle stratégie. Nous avons donc testé l'intérêt de l'utilisation de la molécule pharmacologique AICAR, activateur connu de l'AMPK, afin de « booster » la fonction mitochondriale chez la souris âgée KO mstn. Les résultats montrent chez la souris KO mstn, une amélioration du temps d'endurance de course. Au niveau signalétique, le traitement induit des effets bénéfiques mais limités sur la fonction mitochondriale. Les mécanismes restent à préciser mais tendent vers l'hypothèse d'un effet bénéfique de l'AICAR sur le stress du réticulum endoplasmique (RE). Le dysfonctionnement mitochondrial a été également largement impliqué dans la physiopathologie de la DMD. Dans notre seconde étude, ce même traitement à l'AICAR chez le modèle murin de la DMD, la souris mdx atténue le phénotype dystrophique et améliore la fonction contractile du diaphragme. Nous montrons que ces effets bénéfiques sont associés à une induction de mécanisme de survie, l'autophagie, et une limitation des phénomènes d'apoptose induit par la mitochondrie, mettant en évidence une amélioration de l'intégrité mitochondriale par stimulation de leur renouvellement dans des fibres musculaires dystrophiques. Enfin, ce travail a mis en avant pour la première fois la présence à l'état basal de stress du RE chez la mdx, propsant une nouvelle cible thérapeutique. L'impact de ce stress dans la fibre musculaire normal et pathologique est très mal connu. Nos résultats montrent que le stress du RE modifie les liens entre le réticulum sarcoplasmique et la mitochondrie, perturbe l'homéostasie calcique et active les voies de mort cellulaire associées à une dysfonction contractile. Ces résultats ouvrent une perspective de stratégie thérapeutique dans les pathologies musculaire impliquant un stress du RE, comme la DMD. Ce travail de thèse a mis en avant l'importance de développer des thérapies pharmacologiques dans les pathologies musculaires, permettant d'améliorer la fonction à la fois métabolique et de sentinelle de la mitochondrie. / Fundamental for the energetic balance of the cell, mitochondria play a key role for modulation of cell death pathway related to muscular atrophy. Thus, the purpose of this PhD is to find therapeutic strategy focus on mitochondria in two different murine models where the physiopathology is characterized by a mitochondria dysfunction associated with muscle atrophy: Aging process and Duchenne Muscular Dystrophy (DMD).To prevent loss of muscle mass associated with aging, the lack of myostatin, inducing a hypermuscular phenotype, is a promising therapeutic strategy. However, loss of myostatin is associated with a strong reduction of mitochondrial and oxidative metabolism in skeletal muscle, and this strategy need to be potentiated. In this context, we explore if mitochondrial alteration in aged wild-type mice or in aged mstn KO mice are rescued by chronic AMPK-activating treatment, using the synthetic agonist AICAR, considered as “an mimetic of exercise”. Our results show an improvement of aerobic running performance in mstn KO mice. Concerning to signaling pathways, AICAR treatment induces beneficial but limited effects on mitochondrial metabolism. Mechanisms are still under investigation but our results suggest a reduction in ER stress. Moreover, mitochondria dysfunction has been widely implicated in DMD physiopathology. This same treatment of AICAR, in the murine model of DMD, improves the diaphragm histopathology as well as maximal force generating capacity. These beneficial effects were linked with autophagy activation and apoptosis limitation, without inducing muscle fiber atrophy, and promoting the elimination of defective mitochondria.Finally, the last part of this study highlight for the first time, an increase of ER stress at basal level, suggesting a new therapeutic target. Nevertheless, ER stress impact in skeletal muscle fibers is sparsely known. The preliminary results show that ER stress decrease the link between RE and mitochondria, which have an impact on calcium homeostasis and stimulate cell death pathway with a decrease of contractile function.This study highlights the importance to develop pharmacological therapies in muscular pathology, focus on metabolic and sentinel mitochondria function.

O plexo mioentérico é uma vasta rede de nervos e gânglios localizado entre as camadas longitudinal e circular da túnica muscular externa de todo o trato gastrintestinal (TGI). A distrofia muscular de Duchenne (DMD) é uma miopatia ligada ao cromossomo X causada pela ausência da distrofina que, além dos evidentes efeitos degenerativos no músculo esquelético, causa severas alterações do TGI. No entanto as causas dessas alterações não são claras. Pesquisadores demonstraram que a distrofina é expressa nas fibras musculares lisas e também nos neurônios do plexo mioentérico, todavia não existe um consenso sobre o papel desta nessas estruturas. Desta forma pretende-se estudar os componentes do plexo mioentérico do intestino delgado de camundongos mdx (o modelo animal da DMD) nas idades de 4 e 10 semanas e de seus respectivos controles, camundongos C57BL/10. Os animais de ambos os grupos tiveram o intestino delgado retirado e seccionado em segmentos oral, médio e aboral para posterior avaliação através das técnicas histoquímicas de evidenciação neuronal: NADH-d, NADPH-d e AChE. Além disso, a musculatura lisa e os neurônios do plexo mioentérico foram analisados por MET. A análise quantitativa mostrou que o grupo MDX4 apresentou uma área total do intestino delgado significativamente maior que o C4 (p<0,05). Para as técnicas da NADH-d e NADPH-d foi observado um gradiente crescente de densidade numérica neuronal, no sentido oral-aboral, para todos os grupos estudados. O grupo MDX4 apresentou uma densidade neuronal significativamente menor que o C4 (p<0,05), todavia MDX10 e C10 foram iguais. Além disso, a densidade neuronal dos grupos de 10 semanas foi significativamente menor que dos de 4 semanas para ambas as técnicas (p<0,05). Em relação à estimativa do número total de neurônios, MDX10 e C10 apresentaram uma significativa redução de neurônios NADH-d positivos, quando comparada à dos grupos MDX4 e C4 (p<0,05), porém, para os neurônios NADPH-d positivos, a estimativa dos grupos de 10 semanas foi estatisticamente superior que à dos de 4 semanas (p<0,05). O grupo MDX4 apresentou uma área do perfil de neurônios nitrérgicos significativamente maior que o C4 (p<0,05). Na técnica da NADH-d, não foi detectada diferença significativa relativa à esse aspecto. Comparativamente ao grupo C4, os neurônios do grupo MDX4 não apresentaram intensa reatividade a AChE, mas foram iguais em 10 semanas. Referente à ultra-estrutura dos neurônios, esta apresentou-se preservada em todos os grupos, no entanto as fibras musculares lisas do grupo MDX apresentaram alterações morfológicas. / The myenteric plexus is an extensive network of nerve strands and ganglia located between the outer longitudinal and inner circular muscle layers of the external muscle coat of the gastrointestinal tract (GI). Duchenne muscular dystrophy (DMD) is a X-linked degenerative muscular myopathy caused by the absence of dystrophin which, apart from the obvious degenerative effects in skeletal muscle, causes severe alterations of gastrointestinal (GI) tract. However the causes of these changes remain unclear. Researchers have shown that dystrophin is expressed in both smooth muscle fibers and myenteric plexus neurons, however there is no consensus on the role of it in these structures. Thus, we intend to study the components of the myenteric plexus of the small intestine of mdx mice (an animal model for DMD) at the ages of 4 and 10 weeks and their respective controls, C57BL/10 mice. The animals of both groups had the small intestine removed and sectioned into oral, middle and aboral segments for further evaluation by histochemical techniques of neuronal evidencing: NADH-d, NADPH-d and AChE. In addition, smooth muscle and myenteric plexus neurons were analyzed by TEM. The quantitative analysis showed that the MDX4 group had a significantly higher small intestine total area than the C4 (p <0.05). For the techniques of NADH-d and NADPH-d was observed an increasing gradient of neuronal numerical density, in the oral-aboral direction, for all groups. The MDX4 group showed a significantly lower neuronal density than C4 (p<0.05), however MDX10 and C10 were the same. In addition, the neuronal density of 10 weeks groups was significantly lower than those of 4 weeks for both techniques (p <0.05). Considering the total estimative number of neurons, MDX10 and C10 showed a significant reduction of NADH-d positive neurons, compared to groups MDX4 and C4 (p <0.05), but for the NADPH-d positive neurons, the estimative of 10 weeks groups was statistically higher than that of 4 weeks (p <0.05). The MDX4 group showed an nitrergic neurons profile area significantly higher than the C4 (p <0.05). In the technique of the NADH-d, no significant difference was detected on this aspect. Compared to the C4 group, neurons of MDX4 group did not show intense AChE reactivity, but they were equal in 10 weeks. Concerning the neurons ultrastructure, it was preserved in all groups, however the smooth muscle fibers of MDX group showed morphological changes.

A distrofia muscular de Duchenne (DMD) é uma doença de caráter hereditário recessivo ligado ao cromossomo X, que determina a ausência da distrofia, a responsável pela progressiva degenaração muscular observada no DMD. Embora não expresse o fenótipo, a camundongo mdx apresenta a ausência da distrofia e o mais comum modelo animal experimental para estudar as repercursões da DMD em muitos orgãos. Este estudo foi realizado na cartilagem do processo condilar da mandíbula de mdx, um importante sítio de crescimento craniofacial. Assim, o PC dos mdx de 4 (G1) e 10 (G2) semanas e dos respectivos controles (camundongos c57BL/10 mice) foram avaliados usando as técnicas de microscopia de luz (H.E, Picrosirius e Safranina-O) e de imunohistoquímica (IGF e IGF-IR). Em ambos os grupos, não houve diferença estatísticamente significante na área do PC e comparação aos controles. O número de e a área dos condrócitos, bem como, a quantidade de matriz extracelular (MEC) forma menores nos grupos mdx. A imunorreatividade para ambos, IGF-I e IGF-IR, proporcionalmente maiores nos grupos mdx. Os dados quantitativos e predominância do colágeno tipo I nos grupos mdx, sugere um processo precoce de envelhecimento na cartilagem do PC desses animais. / The duchenne muscular dystrophy (DMD) is a recessive hereditary disease linked to X-chromossome that determines teh dystrophin abstence, the responsible for progressive muscle degeneration observed in DMD. Although not exhibit the phenotype, the MDX mouse reveal abstence of dystrophin and is the most common experimental animal model for DMD studies in many organs. This study was performed in the articular cartilage of the mandibular condylar process (PC) of MDX, an important site of craniofacial growth. Thus the PC of MDX and respective controls (C57BL/10 mice) were evaluated at the ages of 4 (G1) and 10 (G2) weeks using ligth microscopy (H.E, Picrosirius e Safranin-O) and immunohistochemical (IGF-I e IGF-IR) tecniques. In both groups, there was no statistical significant difference in PC area of the mdx and the respective controls. The number and area of the chondrocytes, as well as the amout of extracellular matrix (MEC) were lower in MDX groups. The immunoreactivity for both, IGF-I and IGF-IR, were propostionally higher im MDX groups. The quantitative data and the predominance of collagen type i fibers in the MDX groups suggest a premature aging process of the PC in these animals.

La dystrophie musculaire de Duchenne est une maladie neuromusculaire qui touche 1 enfant sur 3500, liée au chromosome X, caractérisée par l’absence de dystrophine, protéine située sous le sarcolemme qui confère stabilité à la membrane cellulaire en connectant l’actine du cytosquelette avec la matrice extracellulaire. Elle fait partie d’un complexe multi protéique, nommé « dystrophin associated protein complex (DAPC)», qui contient, entre autre, le -dystroglycane et l’oxyde nitrique synthase (NOS). Son absence cause la dérégulation de l’homéostasie calcique, la nécrose tissulaire, l’accumulation de tissu graisseux et fibreux, l’incapacité de mouvement et des déficits cardiaques et respiratoires qui aboutissent au décès des patients. Mon travail avait comme objectif l’amélioration de différents aspects du phénotype dystrophique. J’ai utilisé des molécules capables d’activer deux voies de signalisations (la voie du NO et l’inhibition des histones deacetylase (HDAC)), connues pour induire l’amélioration du phénotype dystrophique chez la souris mdx, modèle de la maladie. Plus particulièrement, j’ai testé chez la souris, deux mode d’administration du butyrate d’arginine (AB), la drogue de référence car déjà utilisée en clinique sur des jeunes patients pour une autre indication, par gavage et par injection intrapéritonéale. J’ai étudié aussi deux nouvelles molécules dérivées du AB, qui pourraient être administrées par voie orale et être efficace à faible dose : le 3-Hydroxybutyrate arginate (ABE) et le N-butyril arginine (ABA). AB, ABE et ABA ont été testés in vitro sur les myotubes de patients dystrophiques et in vivo sur des souris mdx. L’administration orale du AB a les mêmes effets positifs que l’injection intrapéritonéale chez les souris mdx. Ces résultats démontrent que l’administration par voie orale doit être prise en considération lors des futurs essais cliniques. Dans un deuxième temps, je me suis focalisée sur les défauts cardiaques. Un suivi par échocardiographie mensuelle a été réalisé sur des souris de 8 mois traitées avec du AB. En parallèle nous avons analysé les effets de l’administration par voie orale du AB sur les déformations de la colonne vertébrale. Enfin, les altérations des signaux de l’électromyogramme (réalisé avec une méthode non invasive développée en clinique et appliquée pour les animaux) ont été également analysées. L’ensemble des résultats obtenus montre que le AB est capable de préserver l’activité cardiaque, d’empêcher la déformation de la colonne vertébrale et de rétablir les paramètres d’excitabilité axonale mesurés chez les souris traitées.Différentes concentrations des ABE et ABA ont été testé in vivo et observé à des faibles doses les mêmes résultats bénéfiques sur de nombreux paramètres structuraux et fonctionnels, que ceux obtenu avec une dose importante de AB (800mg/kg/j). Les deux nouvelles drogues peuvent être administrées à une dose 10 fois inferieur que la dose de AB pour obtenir les mêmes effets. J’ai testé aussi in vitro, sur des cellules musculaires humaines, la capacité des deux produits à induire une augmentation des niveaux intracellulaires d’utrophine et des protéines associées (β-dystroglycan et la myosine embryonnaire). J’ai aussi démontré qu’une augmentation de l’expression de l’utrophine et des protéines associées pouvait être induite par les inhibiteurs d’HDAC (le butyrate, la trichostatine A, l’acide valproique et l’isobutyramide). Enfin, une étude portant sur l’homéostasie calcique a été réalisé car des altérations de cet équilibre sont en partie responsables de la nécrose/dégénérescence du tissue musculaire. En particulier, l’activité spontanée du Ca2+, enregistrée sur le myotubes humaine, été fortement réduite après un traitement agissant sur la voie d’activation du NO et/ou par des inhibiteurs des HDAC. L’ensemble des résultats obtenus apportent la preuve des effets bénéfiques du AB et de ses dérivés sur la DMD, a travers la voie du NO et en inhibant les HDAC. / Duchenne muscular dystrophy is a X-linked progressive neuromuscular disease affecting 1:3500 boys at birth. It is caused by the absence of dystrophin, a subsarcolemmal protein that confers membrane stability linking cytoskeletal actin to the extracellular matrix. It is part of a multi-protein complex called dystrophin associated protein complex (DAPC), which contains, among the other components, -dystroglycan and nitric oxide synthase (NOS).The consequences of the absence of dystrophin are: deregulation of calcium homeostasis, tissues necrosis, progressive accumulation of fat and fibrosis, inability of the movements and cardiac and respiratory failures that lead to patient’s death, around the age of 20-30 years.The objective of my PhD work is to ameliorate different aspects of dystrophic phenotype. In particular I have tested two different ways of administration of arginine butyrate (AB), the reference drug, through feeding-force and intraperitoneal injection. Meanwhile I have studied two new pharmacological molecules, AB derived, which could be administered orally to DMD patients. These compounds are: 3-Hydroxybutyrate arginate (refer as ABE) and N-butyryl arginine (refer as ABA). All of these molecules partially restore dystrophic phenotype activating two independent pathways (both the nitric oxide pathway and the inhibition of the histone deacetilase), which are known to be beneficent for mdx mice.AB, ABE and ABA have been tested in vitro on human DMD myotubes and in vivo on the mdx mice. The first goal of my project is the observation that the positive effects obtained after intraperitoneal injections of AB can be detected also after oral protocol, promoting the idea that the oral way has to be developed for future clinical trials. I have focused my attention on heart defaults; in particular, starting from the 8th month, a monthly study on heart activity based on echocardiography has been performed on mdx mice treated with AB. We addressed the potential profits of the oral administration of arginine butyrate on vertebral column deformation and electromyogram defaults, with a non-invasive automatized method developed in clinic and then applied to animals. The results collected from these experiments show that AB preserve heart activity, reverse vertebral column deformity and all the axonal excitability parameters that were modified in saline-treated mdx mice.In complement, I have tested different concentrations of ABE and ABA in vivo. The positive effects on many structural and functional dystrophic parameters, previously obtained with high dose of AB administered per os (800 mg/kg/d), has been observed with doses 10 times lower with both new compounds.In parallel, both products were tested in vitro on human muscular cells cultures to investigate their capacity to increase utrophin level. Moreover, the potential ability of histone deacetylase inhibitors (byturate, valproic acid, trichostatin A and isobutyramide) to increase the expression of utrophin and related proteins (-dystroglycan and embryonic myosin) has been studied. Finally, the alteration of calcium homeostasis, largely implicated in the cascades resulting in muscle necrosis/degeneration, was investigated. The spontaneous Ca2+ activity recorded in patient myotubes, i.e. without sarcolemmal integrity was strongly reduced after treatment acting on the NO-pathway activation and/or with HDAC inhibitors. All together, these data constitute a proof of principle of the beneficial effects of arginine butyrate and its derivates on muscular dystrophy, by enhancing NO pathway and inhibiting HDAC.

A distrofia muscular de Duchenne (DMD) é uma doença de caráter hereditário recessivo ligado ao cromossomo X, que determina a ausência da distrofia, a responsável pela progressiva degenaração muscular observada no DMD. Embora não expresse o fenótipo, a camundongo mdx apresenta a ausência da distrofia e o mais comum modelo animal experimental para estudar as repercursões da DMD em muitos orgãos. Este estudo foi realizado na cartilagem do processo condilar da mandíbula de mdx, um importante sítio de crescimento craniofacial. Assim, o PC dos mdx de 4 (G1) e 10 (G2) semanas e dos respectivos controles (camundongos c57BL/10 mice) foram avaliados usando as técnicas de microscopia de luz (H.E, Picrosirius e Safranina-O) e de imunohistoquímica (IGF e IGF-IR). Em ambos os grupos, não houve diferença estatísticamente significante na área do PC e comparação aos controles. O número de e a área dos condrócitos, bem como, a quantidade de matriz extracelular (MEC) forma menores nos grupos mdx. A imunorreatividade para ambos, IGF-I e IGF-IR, proporcionalmente maiores nos grupos mdx. Os dados quantitativos e predominância do colágeno tipo I nos grupos mdx, sugere um processo precoce de envelhecimento na cartilagem do PC desses animais. / The duchenne muscular dystrophy (DMD) is a recessive hereditary disease linked to X-chromossome that determines teh dystrophin abstence, the responsible for progressive muscle degeneration observed in DMD. Although not exhibit the phenotype, the MDX mouse reveal abstence of dystrophin and is the most common experimental animal model for DMD studies in many organs. This study was performed in the articular cartilage of the mandibular condylar process (PC) of MDX, an important site of craniofacial growth. Thus the PC of MDX and respective controls (C57BL/10 mice) were evaluated at the ages of 4 (G1) and 10 (G2) weeks using ligth microscopy (H.E, Picrosirius e Safranin-O) and immunohistochemical (IGF-I e IGF-IR) tecniques. In both groups, there was no statistical significant difference in PC area of the mdx and the respective controls. The number and area of the chondrocytes, as well as the amout of extracellular matrix (MEC) were lower in MDX groups. The immunoreactivity for both, IGF-I and IGF-IR, were propostionally higher im MDX groups. The quantitative data and the predominance of collagen type i fibers in the MDX groups suggest a premature aging process of the PC in these animals.

Orientador: José Angelo Camilli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T23:25:02Z (GMT). No. of bitstreams: 1 Nakagaki_WilsonRomero_D.pdf: 4233267 bytes, checksum: 97a4bebd0547a5885f015f496b45e99f (MD5) Previous issue date: 2011 / Resumo: A distrofia muscular de Duchenne (DMD) é uma doença neuromuscular resultante da ausência de distrofina. Em virtude do enfraquecimento muscular e do uso de glicocorticóides, pacientes com DMD têm ossos frágeis. O camundongo mdx é o modelo experimental largamente utilizado para o estudo da DMD e apresenta falta da distrofina, processo inflamatório intenso e degeneração da fibra muscular. Além disso, apresenta ciclos de degeneração/regeneração muscular que se iniciam de forma mais marcante após o vigésimo primeiro dia de vida. Estudos demonstraram que camundongos mdx têm níveis elevados de fatores de crescimento de fibroblasto e proteína quimiotática de monócito-1, bem como aceleração da cicatrização em lesões da pele. Com base nessas evidências, elaboramos duas hipóteses. A primeira hipótese é que podem existir alterações nos ossos de camundongos mdx por influência da ausência de distrofina ou por algum outro mecanismo inerente à doença mesmo antes da sua manifestação clínica. A segunda hipótese é que o processo de reparo ósseo espontâneo também possa estar acelerado, de modo semelhante à cicatrização da pele. Para testar a primeira hipótese o fêmur e o músculo quadríceps do camundongo mdx foram analisados aos 21 dias de vida. Para verificar a segunda hipótese foi produzido um defeito no osso parietal direito e a regeneração foi analisada após 15, 30 e 60 dias pós-cirúrgicos. Na análise morfológica do quadríceps as fibras musculares apresentavam núcleos periféricos e não foram observadas fibras positivas para o corante azul de Evans em ambos os grupos, indicando que não houve degeneração das fibras no grupo mdx. O fêmur do grupo mdx demonstrou osteopenia, menor quantidade de osteoblastos, menor conteúdo mineral e menor resistência mecânica na ausência de sinais de degeneração muscular em relação ao grupo controle. No estudo do osso parietal, os dados mostraram que não há diferença significante no volume de osso neoformado entre os grupos controle e mdx nos três tempos pós-operatórios e também entre os três tempos, independentemente do grupo estudado. Diante destes resultados, concluímos que o fêmur dos camundongos mdx com 21 dias de vida pode conter um distúrbio interligado a algum fator genético, diretamente ou não relacionado com a ausência de distrofina. Isto demonstrou que a perda da qualidade óssea em camundongos mdx não ocorre somente em função do enfraquecimento muscular. Considerando a qualidade óssea inferior do fêmur e a similaridade estatística da taxa de regeneração óssea, entendemos que a capacidade osteogênica da calvária mdx foi mais expressiva do que a dos camundongos controle, igualando a taxa de reparo ósseo de um tecido com menor qualidade à de ossos normais / Abstract: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by lack of dystrophin. DMD patients have brittle bones because of muscle weakness and use of glucocorticoids. The mdx mouse is widely used as experimental model for the study of DMD and it presents lack of dystrophin, intense inflammatory process and muscle fiber degeneration. Moreover, it presents cycles of muscle degeneration/regeneration that becomes more marked after the twenty-first day of life. Studies have shown that mdx mice have elevated levels of fibroblast growth factor and monocyte chemoattractant protein-1, as well as accelerate wound healing in skin lesions. Based on this evidence, we formulate two hypotheses. The first hypothesis is that there may be changes in the bones of mdx mice by the influence of the absence of dystrophin or by some other mechanism inherent to the disease even before clinical manifestation. The second hypothesis is that the process of spontaneous bone repair can also be accelerated, similar to skin healing. To test the first hypothesis, the femur and the quadriceps muscle of mdx mice were analyzed at 21 days of life. To verify the second hypothesis a defect was produced in the right parietal bone and the regeneration was evaluated after 15, 30 and 60 days after surgery. In the morphological analysis of quadriceps were observed muscle fibers with peripheral nuclei and were not seen Evans blue dye positive fibers in both groups, indicating that there was no fiber degeneration in mdx group. The femur of the mdx group demonstrated osteopenia, lower number of osteoblasts, lower mineral content and lower mechanical strength in the absence of signs of muscular degeneration compared to the control group. In the study of the parietal bone, the data showed no significant difference in newly formed bone volume between control and mdx groups in the three moments after the operation and also between the three moments, regardless of the studied group. Given these results, we conclude that the femur of mdx mice at 21 days of life can contain a disorder linked to some genetic factor, directly or not related to the absence of dystrophin. This demonstrated that loss of bone quality in mdx mice occurs not only because of muscle weakening. Considering the lower femur bone quality and statistical similarity in the rate of bone regeneration, we believed that the osteogenic capacity of mdx calvaria was more expressive than that of control mice, equaling the rate of bone repair of a tissue with lesser quality to that of normal bones / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Orientadores: Maria Julia Marques, Elaine Minatel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T06:25:49Z (GMT). No. of bitstreams: 1 Machado_RafaelVentura_M.pdf: 800859 bytes, checksum: 1bb3eb2604bb7183c771d752b2478081 (MD5) Previous issue date: 2006 / Resumo: Neste trabalho foi verificado se o cromoglicato de sódio protege os músculos distróficos de camundongos mdx da necrose. Camundongos mdx (n=8) com 14 dias de vida pós-natal, antes do início dos ciclos de degeneração e regeneração, foram tratados com cromoglicato de sódio (50mg/Kg/dia; intraperitoneal) por 16 dias. Camundongos mdx (n=8) com a mesma idade foram utilizados como grupo controle, recebendo salina pela mesma via e período. A necrose muscular foi quantificada através do marcador azul de Evans (AE), que penetra na fibra muscular somente quando há lesão do sarcolema. Secções do terço médio dos músculos esternomatóideo e tibial anterior foram obtidas para análise em HE e AE. Foram avaliados o número de fibras musculares positivas ao AE, de fibras musculares com núcleo periférico e de fibras com núcleo central. Foram quantificadas as áreas com infiltrado inflamatório exuberante com células no estágio inicial de regeneração muscular (Área Infl/Reg) e áreas com infiltrado inflamatório escasso com células em estágio avançado de regeneração. O cromoglicato de sódio promoveu diminuição significativa da mionecrose (p<0,05; teste t de Student) em ambos músculos e aumento da porcentagem de fibras com núcleo periférico. No músculo tibial anterior, a diminuição da mionecrose foi de 26% e o aumento de fibras com núcleo periférico, 30%. A área de Infl/Reg aumentou em ambos os músculos (p<0,05; teste t de Student). Os resultados mostram que o cromoglicato de sódio, ministrado antes do início dos ciclos de degeneração/regeneração, protege os músculos distróficos da mionecrose e interfere nos estágios iniciais da regeneração / Abstract: In the present study, we verified whether disodium cromoglycate (cromolyn), an anti-allergic drug, could protect dystrophic mdx muscIe fibers ITom degeneration. Treated mdx mice (n=8; 14 days of age) received daily intraperitoneal iDJections of cromolyn at a dose of 50mglkg body weight in saline, during 15 days. Cromolyn treatment started before the cycIes of muscIe degeneration-regeneration had started. Control non-treated mice (n=8 mdx) were injected with na equivalent amount of saline. For visualization of muscle fiber damage, treated (n=5) and non­ treated mdx (n=5) mice were injected with Evans blue dye (EBD), a marker of sarcolemmal lesion. Cryostat cross-sections of t.he stemomastoid (STN) and tibialis anterior (TA) muscles were stained with HE. The whole cross-sectional area of the muscIes was divided into a regenerated area, a.Tl area of inflammatory celI infiltration/regeneration a.Tld an area of regeneration. The number of regenerated muscle fibers (central nucIeated fibers), fibers with peripheral c.ell nuclei and degenerated fibers (positive to EBD) was counted in the regenerated area. The areas of inflammatory cell infiltrationlregeneration and of regeneration were expressed as a percentage of the total transverse graft area. Cromolyn lead to a significant decrease in myonecrosis and in t.he percentage of central nucleated fibers (p<O.O5; Student's t test). The number of fibers with peripheral nuclei increased in about 30% in t.he TA muscle. The area of inflammation-regeneration increased (p<O.O5; Student's t test) in the cromolyn treated group. These results show that cromolyn treatment before the cycles of muscle fiber degeneration­ regeneration started protects dystrophic muscIe fiber ITom myonecrosis and promotes the earlier stages ofmuscle fiber regeneration / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Orientador: Humberto Santo Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Médicas / Made available in DSpace on 2018-08-13T22:18:59Z (GMT). No. of bitstreams: 1 Oggiam_DaniellaSilva_M.pdf: 1790150 bytes, checksum: f55f529364fdf6fa2bae8c646e2b2d8b (MD5) Previous issue date: 2009 / Resumo: A distrofia Muscular de Duchenne (DMD) é uma miopatia progressiva causada por uma doença autossômica recessiva ligada ao sexo, que acomete crianças do sexo masculino, e evolui para incapacidade motora na puberdade até causar óbito ao redor da segunda década de vida. É causada por uma alteração no gene codificador da proteína distrofina, que mantém a integridade do sarcolema da fibra muscular. O camundongo mdx é utilizado como modelo experimental da DMD para investigações do tecido muscular esquelético e cardíaco, por apresentar muitas semelhanças com humano portador da doença. Na DMD os pacientes iam á óbito por falência respiratória, desde a evolução do tratamento com técnicas de ventilação mecânica artificial, as disfunções cardíacas tornaram-se importantes, visto que a partir disto, a maioria dos óbitos começaram a ser em função da falência cardíaca, que resulta de um processo seguido de: necrose, inflamação, fibrose evoluindo para cardiomiopatia grave. Várias estratégicas farmacológicas tem sido utilizadas para melhora da função cardíaca tanto no portador de DMD como no camundongo mdx. Um dos medicamentos utilizados é o deflazacorte, um glucocorticóide de ação anti-inflamatória, administrado por toda vida do portador de DMD. Embora os efeitos do deflazacorte sobre a função cardíaca no humano e no camundongo mdx têm sido extensivamente estudados, pouco se sabe sobre os efeitos histopatológicos no tecido cardíaco. Neste trabalho foi avaliado o efeito da administração a longo prazo de deflazacorte na progressão da fibrose miocárdica intersticial em camundongos mdx de 6 meses de idade. Os animais foram tratados diariamente com deflazacorte durante 15 meses, após foram sacrificados, o coração foi removido e congelado em nitrogênio líquido para posterior análise histológica e morfológica do tecido. O coração do grupo de camundongos mdx tratados com deflazacorte foi comparado com camundongos mdx não tratados. As áreas de fibrose miocárdica diminuíram significativamente 40% em relação ao grupo não tratado. Concluiu-se que o tratamento à longo prazo com deflazacorte é eficiente para diminuir a progressão da fibrose cardíaca. Sendo assim, como a cardiomiopatia está diretamente relacionada à disfunções celulares que acarreta a necrose dos cardiomiócitos, é de interesse investigar o acúmulo de lipofuscina, um biomarcador do envelhecimento, nos corações de camundongos mdx. Neste trabalho também foi observado o acúmulo de lipofuscina em animais controle C57BL10 e mdx de 14 dias a 23 meses de idade sem serem submetidos a qualquer tratamento. Os animais foram sacrificados, o coração removido e congelado em nitrogênio líquido para posterior análise da fluorescência dos grânulos de lipofuscina. Após contagem dos grânulos observou-se que aumentam com a idade, e dos 4 para os 6 meses ocorreu um acréscimo no acúmulo de lipofuscina. Considerando-se que o acúmulo de lipofuscina relaciona-se a disfunção celular é possível que isto contribua para lesão de cardiomiócitos em corações desprovidos de distrofina / Abstract: The Duchenne Muscle Dystrophy is a progressive myopathy caused by recessive autossomic disease connected to the gender, which attacks male kids, and involves to motor disability in the property, leading to death around the second decade of life. It is caused by an alteration in the codifier gene of the protein dystropin, which maintains the integrity of the muscle fiber sarcolemma. The mdx mouse is used as an experimental model of DMD to investigate the skeletal and cardiac muscle fiber, because it presents a lot of similarities with the human carrier of the disease. In the DMD, the patients used to die due to respiratory failure. Since there was a treatment evolution with artificial mechanical ventilation techniques, the cardiac dysfunctions became important considering that from this moment on, most of the deaths started occurring because of a cardiac failure, resulting of a process followed by necrosis, inflammation, fibrosis involving to a serious cardiomyopathy. Several pharmacological strategies have been used to improve the cardiac function both in the DMD carrier and in the mdx one of the medications utilized is the deflazacort, a glucocorticóide of anti-inflammatory action, administrated during the whole life of the DMD carrier. Although the deflazacort effects upon the cardiac function in the human being and in the mouse mdx have been extensively studied just a little is know about the histopathological effects on the cardiac tissue. In this paper, the effect of the long term administration of deflazacort daily for 15 months, after they were sacrificed, had their hearts removed and frozen in liquid nitrogen for histological and morphological tissue further analysis. The heart of the mdx mice group treated with deflazacort was compared to the heart of the untreated mdx mice group. The myocardial fibrosis areas diminished significantly in comparison to the untreated group, in 40%. It was concluded that the long term treatment with deflazacort is effective to diminish the cardiac fibrosis progression the cardiomyopathy which cause the myocites cardiac necrosis, and therefore it is interesting to investigate the lipofuscin is a pigment related to the age, it is considered an aging biomarker. In this paper, the accumulation of lipofuscin in control animals C57BL10 and mdx with ages between 14 days and 21 months without any treatment was observed that they increase with the age, and from the 4 to the 6 months there was a raise in the lipofuscin accumulation. It was so, concluded, that the myocites cardiac functioning can be harmed even before the age of 8 months, and the accumulation of lipofuscin can mean a degeneration process which is more intensive in mdx / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T07:04:11Z (GMT). No. of bitstreams: 1 Albuquerque_TerezaCristinaPessoade_M.pdf: 1316775 bytes, checksum: 93039fe90ce98e8b3c47f1e5074f823b (MD5) Previous issue date: 2008 / Resumo: Em fibras musculares distróficas de camundongos mdx e na distrofia muscular de Duchenne (DMD), a deficiência da distrofina provoca lesão do sarcolema e degeneração muscular. Esta deficiência está associada a alterações na estabilidade do sarcolema e aumento dos níveis intracelulares de cálcio, que podem provocar mionecrose. O mecanismo exato que provoca a lesão da membrana e a mionecrose são desconhecidos. Uma das hipóteses é que a resposta inflamatória endógena pode aumentar a lesão do sarcolema devido à ausência da distrofina. O processo inflamatório está associado com a regeneração muscular e com o reparo tecidual. Antiinflamatórios não-esteróides (AINE) podem reduzir a inflamação que ocorre como conseqüência de lesão muscular através da inibição das ciclo-oxigenases, provocando redução da síntese de prostaglandinas. Estudos anteriores mostraram que houve atraso da regeneração muscular após a utilização de AINE. Neste estudo, foi verificado se os AINE naproxeno e nimesulida reduzem a mionecrose no camundongo mdx, um dos modelos experimentais da DMD. Camundongos mdx (n= 48) com 21 dias de idade receberam injeções intraperitoneais de naproxeno (10 mg/kg de peso corporal) ou de nimesulida (25 mg/kg de peso corporal) durante 15 ou 30 dias consecutivos. Camundongos mdx não tratados receberam injeção intraperitoneal de solução salina. Para a observação e contagem de fibras em degeneração, três animais de cada grupo receberam injeção intraperitoneal de azul de Evans (AE), marcador que indica alterações de permeabilidade do sarcolema. Secções transversais dos músculos esternomastóide (STN) e tibial anterior (TA) foram coradas com hematoxilina-eosina. A secção total transversa foi dividida em área de infiltrado inflamatório, com fibras em estágios iniciais de regeneração (áreas INFL/REG), e áreas com fibras em estágio avançado de regeneração (áreas REG). Estas áreas foram expressas como porcentagens da área seccional transversa total. O número de fibras regeneradas (fibras com núcleo central), fibras com núcleo periférico e fibras em degeneração (positivas ao corante AE) foram quantificadas. O naproxeno mostrou-se mais efetivo que a nimesulida na diminuição da mionecrose, influenciando de maneira diferenciada os ciclos de degeneração/regeneração de músculos distróficos, principalmente quando utilizado nos estágios iniciais destes ciclos. Concluímos que a utilização de inibidores da produção de prostaglandinas não prejudica a regeneração muscular de camundongos mdx, podendo ser uma alternativa para o tratamento de miopatias, especialmente se associada a outros fármacos de ação mais direta na proteção à mionecrose / Abstract: In dystrophin-deficient fibers of mdx mice and in Duchenne muscular dystrophy (DMD), the lack of dystrophin leads to sarcolemma breakdown and muscle degeneration. The lack of dystrophin is associated with changes in membrane stability and increased levels of calcium in the muscle fiber, which leads to myonecrosis. The exact mechanism determining the sarcolemmal lesion and myonecrosis is unknown. One hypothesis is that the endogenous inflammatory response exacerbates the muscle fiber membrane damage due to the lack of dystrophin. Inflammation has also been associated to muscle regeneration and repair. Non-steroidal anti-inflammatory drugs (NSAID) can reduce inflammation following injury by inhibiting cyclooxygenase and leading to a reduction in prostaglandin synthesis. Previous experimental studies have indicated delayed muscle regeneration after NSAID. In this work, we verified the effects of the cyclooxygenase inhibitors, naproxen and nimesulide, on the extent of myofiber necrosis in the mdx mice model of DMD. Mdx mice (n=48) at 21 days after birth received daily intraperitoneal injections of naproxen (10 mg/kg body weight) or nimesulide (25 mg/kg body weight) for 15 or 30 days. Untreated mdx mice were injected with saline. To measure muscle fiber damage, some mice were injected with Evans blue dye (EBD; n=3), a marker of sarcolemmal lesion. Cryostat cross-sections of the sternomastoid (STN) and tibialis anterior (TA) muscles were stained with hematoxylin and eosin. The whole cross-sectional area of the muscles was divided into areas of inflammatory infiltrate with early regenerating fibers (INFL/REG areas) and areas with late regenerated fibers (REG areas). These areas were expressed as a percentage of the total cross-section area. The number of regenerated fibers (central nucleated fibers), fibers with peripheral cell nuclei and degenerated fibers (positive to EBD) was counted. Naproxen was more effective than nimesulide in decreasing myonecrosis. We concluded that the use of prostaglandin inhibitors does not impair muscle regeneration in the mdx mice. They could be useful therapeutic alternatives in the treatment of DMD, but should be accompanied by other strategies directed against muscle degeneration / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural