Distribuição dos receptores de acetilcolina e da oxido nitrico sintase neuronal na junção neuromuscular de fibras musculares regeneradas

Orientador: Maria Julia MarquesTese(mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O objetivo do presente trabalho foi investigar se fibras musculares regeneradas de camundongos normais apresentam alteração no padrão de distribuição dos receptores de acetilcolina (AChRs) e na expressão da óxido nítrico sintase neuronal (nNOS) e comparar os resultados obtidos com fibras regeneradas de camundongos mdx. Para tanto, a degeneração/regeneração foi induzida através de injeções de cloridrato de lidocaína no músculo esternomastóideo (STN) esquerdo de camundongos normais. O lado contralateral foi utilizado como controle. Após 21 dias os músculos foram retirados e marcados com rodamina-a-bungarotoxina para observação dos AChRs, com o anticorpo primário monoclonal anti-nNOS para observação da expressão da nNOS e montados para tluorêscencia. Foi adicionado ao meio de montagem o marcador de DNA nuclear DAPI o que possibilitou a observação dos núcleos celulares. Através da microscopia confocal de tluorêscencia observamos que a distribuição dos AChRs nas fibras regeneradas estava alterada em relação ao padrão das fibras do músculo controle. As fibras musculares regeneradas normais exibiram o mesmo padrão de distribuição dos AChRs observado em fibras regeneradas distróficas, ou seja, os braços juncionais estavam fragmentados, originando ilhas de receptores. Nossos resultados sugerem que a alteração no padrão de distribuição dos AChRs, que é observada no camundongo mdx, pode ser conseqüência apenas da degeneração/regeneração da fibra muscular e não devido a deficiência de distrofina. Quanto a nNOS, não houve alteração de sua expressão em fibras musculares regeneradas quando comparada com fibras do músculo controle. Isto sugere que a diminuição de sua expressão observada nos camundongos mdx pode ser atribuída a ausência de distrofinaAbstract: The aim of the present investigation was to study whether normal regenerated muscle fibers show changes in the pattem of distribution of the acetylcholine receptors (AChRs) and in neuronal nitric oxide synthase (nNOS) expression. Degenerative-regenerative changes were experimentaly induced in normal muscle fibers of mouse left sternomastoid (STN) muscle by injecting lidocaine hydrochIoride. The contralateral side .served as a controI. 21 days after lidocaine injection, the STNs (right and left) were removed, labelled with rhodamin a-bungarotoxin for AChRs observation, with anti-nNOS for nNOS observation and mounted in special mouting media for fluorescence with DAPI, a DNA stain for nuclei. By using fluorescence confocal microscopy regenerated muscle fibers showed disrupted AChRs distribution when compared to controls. Normal regenerated fibers exhibited the same pattem of AChR distribution as seen in dystrophic regenerated fibers. Our results suggest that changes in AChRs distribution seen in mdx were probably a consequence of muscle fiber degeneration-regeneration, rather than a symptom of dystrophin deficiency. Conversely, there were no changes in nNOS expression in normal regenerated fibers when compared to normal fibers, suggesting that the decrease in nNOS expression reported for mdx mice might be attributed to the absence of dystrophinMestradoAnatomiaDoutor em Biologia Celular e Estrutura

Distribuição dos receptores de acetilcolina e eliminação sinaptica durante o desenvolvimento da junção neuromuscular do camundongo MDX

Orientador: Maria Julia MarquesTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As junções neuromusculares (JNM) dos vertebrados, inicialmente polinervadas, tomam-se monoinervadas em um processo denominado eliminação sináptica. Os mecanismos envolvidos neste processo ainda são pouco conhecidos, sendo uma das possibilidades a de que mudanças no nível das moléculas da membrana pós-sináptica poderiam levar a remoção dos terminais nervosos em excesso. Neste sentido, a JNM distrófica se toma um modelo interessante para o estudo dos mecanismos envolvidos na eliminação sináptica, uma vez que as fibras distróficas não expressam distrofina e apresentam redução de moléculas do complexo distrofina-glicoproteina, complexo este que faz parte do citoesqueleto pós-sináptico e que possivelmente está envolvido na estabilização dos receptores de acetilcolina (AChRs). Desta forma, no presente trabalho verificamos se a eliminação sináptica e a distribuição dos receptores de acetilcolina, observados durante o desenvolvimento pós-natal, estão alterados na fibra deficiente de distrofina. Os AChRs e os terminais nervosos do músculo estemomastóide de camundongos mdx e controle (C57BL/1O) foram marcados, respectivamente, com rodamina-a.-bungarotoxina e anticorpo anti-neurofilamento. Através da microscopia confocal, observamos que 7 dias após o nascimento a maioria das JNMs do camundongo mdx se encontravam monoinervadas (86,7% - n=200), enquanto a mesma observação foi feita em 41,4% no controle (n=200). Consistente com este fato, no mesmo período, a presença de placas perfuradas é mais fTequente no mdx (18,6%) do que no controle (7,3%). No final da segunda semana pósnatal, todas as JNMs se encontravam monoinervadas (100% mdx e 94,7% controle, n=200 para cada grupo) e os AChRs apresentavam o padrão de distribuição adulto. No camundongo mdx, a presença de AChRs distribuídos em pequenas ilhas foi observada no período de 21 dias pós-natal em 13,3% das JNM, indicando a presença de fibras musculares regeneradas. Nossos resultados demonstram que a eliminação sináptica ocorre mais cedo nas fibras musculares distróficas quando comparada ao de um animal controle, sugerindo assim que a estabilização da fibra muscular pela distrofina ou pelo complexo distrofinaglicoproteínas normal é necessário para o curso da eliminação sináptica em tempo normalAbstract: The vertebrate neuromuscular junction goes trom multiple to monoinervated during early postnatal life in a process calIed synapse elimination. The mechanisms underlying this process are unknown but one possibility would be that changes at in the molecular level in the postsynaptic cell lead to the removal of the overlying nerve terminal. The mdx mice show a deficiency of dystrophin and associated proteins, which are part of the postsynaptic cytoskeleton and possibly involved in acetylcholine receptors (AChRs) stabilization. In the study, we used rhodamine-a-bungarotoxin and anti-neurofilament-IgG-FITC to stain AChRs and nerve terminals of the sternomastoid muscle, during early postnatal development of mdx and control C57BL/lO mice. Using fluorescence confocal microscopy, we observed that 7 days after birth, 86,7% of the mdx endplates were monoinervated (n=200) while the same observation was made in 41,4% of the controls (n=200). Consistent with this is the fact that, at this time, perforated plaques were seen more trequent1y in mdx (18,6%) than controls (7,3%). By the end of the second postnatal week, all the endplates were monoinervated (100% mdx and 94,7% controls, n=200 for each group) and AchRs had attained the branched-pattern distribution of adults. In mdx mice, breaking down of receptors into islands, as observed in adult mdx, were seen 21 days after birth in 13,3% of the junctions, indicating the presence of regenerated muscle fibers. These results show that synapse elimination takes place earlier in dystrophin-deficient muscle fibers of the mdx mice, suggesting that stabilization of muscle fiber by dystrophin or a nonnal cytoskeleton complex is required for the nonnal time course of synapse eliminationDoutoradoAnatomiaDoutor em Biologia Celular e Estrutura

Nerve-terminal And Schwann-cell Response After Nerve Injury In The Absence Of Nitric Oxide.

Dystrophic muscles show alterations in the dystrophin-glycoprotein complex and a lack of neuronal nitric oxide (NO) synthase. In mdx mice, presynaptic expression of neuronal NO synthase is decreased, suggesting that presynaptic signaling may be altered in dystrophic muscle. In this study, we examined the nerve-terminal and Schwann-cell responses after a crush lesion in control and NO-deficient mice. Seven days after nerve crush, 24% of control neuromuscular junctions (n = 200) showed ultraterminal sprouts, whereas in NO-deficient mice this frequency was 28.5% (n = 217; P > 0.05 compared to controls; chi-square test). Schwann-cell response did not change in the absence of NO, after a nerve lesion of 7-day duration. Fourteen days after the lesion, nerve terminals sprouted and Schwann cells showed an extensive network of processes away from the synaptic site in controls. In the absence of NO, there was a dramatic decrease in nerve-terminal sprouting and Schwann-cell processes failed to extend away from the endplate. These results show that NO is involved in the nerve-terminal and Schwann-cell response to nerve injury. They also suggest that presynaptic molecular signaling may be impaired in dystrophic muscles, and this could influence the innervation and survival of newly formed myofibers generated by cell-mediated therapies.34225-3

Understanding The Beneficial Effects Of Doxycycline On The Dystrophic Phenotype Of The Mdx Mouse.

The purpose of this study was to better understand the beneficial effects of doxycycline on the dystrophic muscles of the mdx mouse. Doxycycline (DOX) was administered for 36 days, starting on postnatal day 0, via drinking water. Untreated mdx mice received plain water for the same period and served as a control group. DOX decreased the levels of metalloproteinase-9 and tumor necrosis factor-alpha in the biceps brachii and diaphragm of the mdx mice. It also reduced the total amount of calcium in the muscles studied, concomitant with an increase in the levels of calsequestrin 1. The results show that DOX can affect factors that are important in dystrophic pathogenesis and highlight its potential as a readily accessible therapy in clinical trials for treatment of Duchenne muscular dystrophy.50283-

Intrinsic Laryngeal Muscles Are Spared From Myonecrosis In The Mdx Mouse Model Of Duchenne Muscular Dystrophy.

Intrinsic laryngeal muscles share many anatomical and physiological properties with extraocular muscles, which are unaffected in both Duchenne muscular dystrophy and mdx mice. We hypothesized that intrinsic laryngeal muscles are spared from myonecrosis in mdx mice and may serve as an additional tool to understand the mechanisms of muscle sparing in dystrophinopathy. Intrinsic laryngeal muscles and tibialis anterior (TA) muscle of adult and aged mdx and control C57Bl/10 mice were investigated. The percentage of central nucleated fibers, as a sign of muscle fibers that had undergone injury and regeneration, and myofiber labeling with Evans blue dye, as a marker of myofiber damage, were studied. Except for the cricothyroid muscle, none of the intrinsic laryngeal muscles from adult and old mdx mice showed signs of myofiber damage or Evans blue dye labeling, and all appeared to be normal. Central nucleation was readily visible in the TA of the same mdx mice. A significant increase in the percentage of central nucleated fibers was observed in adult cricothyroid muscle compared to the other intrinsic laryngeal muscles, which worsened with age. Thus, we have shown that the intrinsic laryngeal muscles are spared from the lack of dystrophin and may serve as a useful model to study the mechanisms of muscle sparing in dystrophinopathy.35349-5

Nerve Terminal Contributes To Acetylcholine Receptor Organization At The Dystrophic Neuromuscular Junction Of Mdx Mice.

Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1-month-old) and adult (6-month-old) mdx mice was injected with 60 microl lidocaine hydrochloride to induce muscle degeneration-regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4-Di-2-ASP and rhodamine-alpha-bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated-regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated-regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve-dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution.290181-

Understanding the beneficial effects of doxycycline on the dystrophic phenotype of the mdx mouse

Introduction: The purpose of this study was to better understand the beneficial effects of doxycycline on the dystrophic muscles of the mdx mouse. Methods: Doxycycline (DOX) was administered for 36 days, starting on postnatal day 0, via drinking water. Untreated mdx mice received plain water for the same period and served as a control group. Results: DOX decreased the levels of metalloproteinase‐9 and tumor necrosis factor‐alpha in the biceps brachii and diaphragm of the mdx mice. It also reduced the total amount of calcium in the muscles studied, concomitant with an increase in the levels of calsequestrin 1. Conclusions: The results show that DOX can affect factors that are important in dystrophic pathogenesis and highlight its potential as a readily accessible therapy in clinical trials for treatment of Duchenne muscular dystrophy502283286CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP301306/2010‐9; 302006/2009‐5; 474708/06‐304/15526‐9; 08/58491‐1; 11/51697‐6; 142935/2011‐5; 08/54775‐

Ascorbic acid protects the diaphragm muscle against myonecrosis in mdx mice

Objective: Oxidative stress contributes to myonecrosis in the dystrophin-deficient fibers of mdx mice and in Duchenne's muscular dystrophy. We examined the effects of ascorbic acid (AA), an antioxidant and free radical scavenger, on the dystrophic diaphragm muscle. Methods: Mdx mice (14 d old) received AA for 14 d. Control mdx mice received saline. The muscle damage was visualized by the penetration of Evans blue dye into myofibers and the extent of inflammation was assessed by histologic analysis. Creatine kinase levels were measured for the biochemical evaluation of muscle fiber degeneration. The levels of tumor necrosis factor-alpha (a proinflammatory cytokine) and 4-hydroxynonenal (a marker of lipid peroxidation) were analyzed by immunoblotting. Results: Ascorbic acid decreased creatine kinase levels, myonecrosis, inflammation, and the levels of tumor necrosis factor-alpha and 4-hydroxynonenal. Conclusion: The present results suggest that AA plays a protective role in dystrophic muscle degeneration, possibly by decreasing reactive oxygen species, and support further investigations of AA as a potential therapy for dystrophinopathies286686690CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302006/09-5; 301386/07-2sem informaçãosem informaçã