Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (106 cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodaminelabeled alpha-bungarotoxin. the development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. the presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 +/- 1.0 mu m; N = 213), the diameter of ectopic fibers (59.1 mu m; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.UNIFESP, EPM, Dept Farmacol, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Neurol & Neurocirurgia, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Farmacol, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Neurol & Neurocirurgia, BR-04044020 São Paulo, BrazilWeb of Scienc

Distrofia muscular de Duchenne: imunoexpressão da alfa-distroglicana em musculatura esquelética e performance cognitiva

The Duchenne muscular systrophy (DMD) is a muscular dystrophy with cognitive impairment present in 20-30% of the cases. In the present study, in order to study the relationship between the alpha-dystroglycan (alpha-DG) immunostaining in skeletal muscle and cognitive performance in DMD patients, 19 were assessed. Twelve patients performed the intelligence quotient (IQ) below the average. Among the 19 patients, two were assessed by the Stanford-Binet test and 17 by Wechsler Intelligence Scale for Children-III (WISC-III). Nine patients performed a verbal IQ below the average, only three patients performed an average verbal IQ. The muscle biopsies immunostained with antibodies to alpha-DG showed that 17 patients presented a low expression, below 25% of the total fibers. Two patients presented alpha-DG immunostaining above 40% and an IQ within the average. No significant statistical relationship was demonstrated among total IQ, verbal IQ and execution IQ and alpha-DG immunostaining at these patients muscle samples.A distrofia muscular de Duchenne (DMD) é uma distrofia muscular com comprometimento cognitivo presente em 20-30% dos casos. No presente estudo, com a finalidade de estudar a relação entre a imunoexpressão da alfa-distroglicana (alfa-DG) em musculatura esquelética e a performance cognitiva em pacientes com DMD, foram avaliadas 19 crianças. Doze pacientes apresentaram o quociente de inteligência (QI) abaixo da média. Entre os 19 pacientes, dois foram avaliados pelo teste de Stanford-Binet e 17 pelo Wechsler Intelligence Scale para crianças-III (WISC-III). Nove apresentaram QI verbal abaixo da média, e apenas três QI verbal na média. As biopsias musculares com os anticorpos para alfa-DG mostraram que 17 pacientes apresentaram baixa expressão, abaixo de 25% do total de fibras. Dois pacientes apresentaram a imunoexpressão da alfa-DG acima de 40% e QI dentro da média. Não foi demonstrada relação estatisticamente significante entre o QI total, QI verbal e QI de execução e a imunoexpressão da alfa-DG .Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Department of NeurologyCeT Codificação e TabulaçãoUNIFESP, EPM, Department of NeurologySciEL

Nitric Oxide Synthesis Is Increased in Cybrid Cells with m.3243A > G Mutation

Nitric oxide (NO) is a free radical and a signaling molecule in several pathways, produced by nitric oxide synthase (NOS) from the conversion of L-arginine to citrulline. Supplementation of L-arginine has been used to treat MELAS (mitochondrial encephalopathy with lactic acidosis and stroke like syndrome), a mitochondrial disease caused by the m. 3243A>G mutation. Low levels of serum arginine and endothelium dysfunction have been reported in MELAS and this treatment may increase NO in endothelial cells and promote vasodilation, decreasing cerebral ischemia and strokes. Although clinical benefits have been reported, little is known about NO synthesis in MELAS. in this study we found that osteosarcoma derived cybrid cells with high levels of m. 3243A>G had increased nitrite, an NO metabolite, and increased intracellular NO, demonstrated by an NO fluorescent probe (DAF-FM). Muscle vessels from patients with the same mutation had increased staining in NADPH diaphorase, suggestive of increased NOS. These results indicate increased production of NO in cells harboring the m. 3243A>G, however no nitrated protein was detected by Western blotting. Further studies are necessary to clarify the exact mechanisms of L-arginine effect to determine the appropriate clinical use of this drug therapy.Fundacao de Amparo a Pesquisa de São PauloCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Escola Paulista Med, Dept Neurol & Neurosurg, BR-04039032 São Paulo, BrazilUniv Miami, Miller Sch Med, Dept Neurol & Cell Biol, Miami, FL 33101 USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Neurol & Neurosurg, BR-04039032 São Paulo, BrazilWeb of Scienc

Congenital muscular dystrophy: a clinical report on 17 patients

We concur with the idea that congenital muscular dystrophy (CMD) is a distinct clinical entity, and report 17 patients (2 negroes and 15 whites; 12M and 5 F; median age 6 years, range 1 to 24 years) with genetic, clinical, laboratorial, electrophysiological and histochemical studies. All our cases have an inheritance compatible with an autosomal recessive pattern. A decrease in fetal movements was reported by 57% of the mothers, generalized hypotonia at birth was present in 82%, limb girdle and neck weakness, absent or decreased deep tendon reflexes, and limb contractures were present in all. Severe muscular wasting was found in 41%. Calf pseudo-hypertrophy was observed in one patient. A patient was severely mentally retarded and another was borderline. During a 30-month follow-up, the muscle weakness of the majority remained essentially unchanged but the degree of motor activity deteriorated and was proportional to the worsening of the limb contractures. Serum CK levels were normal or increased to a maximum of 8 times. The electromyogram was myopathic in 74%, neurogenic in 13% and normal in 13%. CT scans showed a symmetrical white matter hipodensity in the hemispheres in 8 cases. All but 5 patients were operated upon to release the limb contractures and all were submitted to physical therapy. The contractures recurred in 4 patients submitted to surgery and were probably related to the cessation of physical therapy.Descrevemos 17 pacientes (12m, 5f) com idades que variaram de 1 a 24 anos (mediana 6 anos) com distrofia muscular congênita (DMC), que foram estudados do ponto de vista genético, clínico, laboratorial, eletrofisiológico e anátomo-patológico. A apresentação segundo a herança foi da forma esporádica (76,5%) ou possivelmente autossômica recessiva (23,5%). A diminuição da movimentação fetal intra-uterina foi referida em 57% dos casos, hipotonia neonatal em 82% e retardo no desenvolvimento motor em 88,2%. Fraqueza muscular, diminuição dos reflexos profundos e contraturas articulares estavam presentes em todos os casos. A piora na função motora estava muito relacionada ao aumento ou aparecimento de novas retrações articulares. A CK nunca ultrapassou valores acima de 8 vezes o normal. O ENMG foi de padrão miopático em 73,3%, neuropático em 13,3% e normal em 13,3% dos casos. Aspectos tomográficos com hipodensidade da substância branca subcortical foram vistos em 8 casos. Ao tratamento impôs-se fisioterapia adequada e cirurgia corretiva das deformidades articulares. Novas contraturas desenvolveram-se mais tarde e estavam relacionadas freqüentemente a fisioterapia insuficiente.Escola Paulista de MedicinaUNIFESP, EPMSciEL

Periodic paralysis: anatomo-pathology of skeletal muscle of 14 patients

Periodic paralysis is a rare disease, characterized by transient weakness associated with abnormal levels of serum potassium. Muscle biopsy may show a wide range of abnormalities, vacuoles being more specifically linked to the disease. We analysed 17 muscle biopsies from 14 patients with periodic paralysis (14 hypokalemic, 2 hyperkalemic). All of them showed at least one histological abnormality. Fourteen specimens showed vacuoles that were peripheral, single, frequent and preferentially found in type I fibers. Frequency or severity of attacks did not correlate with the presence of vacuoles but those were more easily found in patients with long term disease. Ten biopsies showed tubular aggregates, specially on the patients with frequent crises or long term disease. A second biopsy was done in three patients and in two we observed a worsening of the histopathologic picture. One patient manifested interictal weakness with evident myopathic changes on the muscle biopsy. Nonspecific changes were found in variable degrees in 15 biopsies. Our study shows that vacuoles and tubular aggregates are frequent changes in periodic paralysis and therefore helpful for the diagnosis. Important myopathic findings in the muscle biopsy suggest a permanent myopathy which probably develops after severe crises or long term disease.A paralisia periódica é entidade caracterizada por crises de fraqueza muscular relacionadas com alterações do nível sérico de potássio. A biópsia muscular pode mostrar alterações específicas ou inespecíficas. Nosso estudo tem como objetivo a análise de 17 biópsias musculares de 14 pacientes com paralisia periódica (14 hipocalêmica, 2 hipercalêmica). Todas as biópsias mostraram alguma alteração histopatológica. Quatorze biópsias apresentavam vacúolos, que se caracterizavam por serem únicos, de localização periférica, de aparecimento frequente e preferentemente em fibras do tipo I. Os vacúolos eram mais visualizados naqueles pacientes com longa evolução e sem relação com a frequência de crises. Os agregados tubulares foram encontrados em 10 biópsias principalmente naqueles pacientes com crises frequentes e doença de longa evolução. Em 3 pacientes foram realizadas 2 biópsias, notando-se piora das alterações em 2. Um paciente evoluiu com quadro clínico de miopatia permanente, confirmado pela biópsia muscular. Alterações inespecíficas foram encontradas em graus variáveis em 15 biópsias. Nosso estudo mostra que os vacúolos e os agregados tubulares são achados frequentes na paralisia periódica, constituindo importante auxílio diagnóstico. Alterações miopáticas evidentes à biópsia sugerem o aparecimento de miopatia permanente, quadro decorrente de doença de longa evolução ou crises severas.Escola Paulista de MedicinaEscola Paulista de Medicina Departamento de Anatomia PatológicaUNIFESP, EPM, Depto. de Anatomia PatológicaSciEL

Frequency of duplications in the D-loop in patients with mitochondrial DNA deletions

Small duplications (miniduplications) of the D-loop of human mitochondrial DNA (mtDNA) have been described in patients with mtDNA deletions, mtDNA point mutations and in normal aged tissues. the origin of these miniduplications is still unknown but it is hypothesized that they could be formed after oxidative damage. the respiratory chain (RC) is the main source of free radicals in mitochondria and it is believed that a defect in RC increases free radical generation. If miniduplications are originated by oxidative damage, it is expected that they are more abundant in patients with a defect in the RC. We studied the frequency of miniduplications of D-loop in patients with a RC defect due to mtDNA deletions and in controls. We show that four types of miniduplications could be detected with a higher prevalence than in previous studies and that patients with mtDNA deletions did not have higher proportions or increased number of miniduplications, which is against the hypothesis that miniduplications are generated more abundantly in patients with RC defects. We also clearly demonstrate the age-related nature of these miniduplications by a carefully controlled study regarding the age of subjects, which was not considered in other studies on patients with a mitochondrial disease. (C) 2002 Elsevier Science B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Disciplina Neurol Clin, Dept Neurol & Neurosurg,Div Neurol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Disciplina Neurol Clin, Dept Neurol & Neurosurg,Div Neurol, BR-04039032 São Paulo, BrazilWeb of Scienc

Miopatia mitocondrial: apresentação de doze pacientes

BV UNIFESP: Teses e dissertaçõe

Mutações do DNA mitocondrial na oftalmoplegia externa progressiva crônica: estudo clínico, molecular e morfológico em biopsias musculares

A oftalmoplegia externa progressiva cronica (OEPC) e uma das manifestacoes mais comuns das doencas mitocondriais. Diversos defeitos do DNA mitocondrial foram identificados na OEPC: delecao simples, delecoes multiplas, mutacoes de ponto e duplicacoes. O principal interesse no presente trabalho foi a deteccao e a caracterizacao das mutacoes do DNA mitocondrial (DNAmit), tentando estabelecer uma correlacao com as alteracoes morfologicas encontradas em biopsias musculares e com a gravidade clinica, em 15 pacientes com a OEPC. Para isso, as biopsias musculares foram estudadas atraves de tecnicas histoquimicas, imuno-histoquimicas e analise do DNAmit. As delecoes do DNAmit foram mapeadas e quantificadas por Southern blotting, reacao em cadeia da polimerase e sequenciamento. Nove pacientes apresentaram delecoes simples heteroplasmicas, de 2309 a 7566 pares de bases (bp): tres deles tinham a mesma odelecao comumode 4977 bp. A proporcao do DNAmit deletado variou de 14 a 89%. Os limites das delecoes foram sequenciados em 8 pacientes e todos apresentaram repeticoes diretas de 8 a 13 bp. Os genes deletados codificavam subunidades dos complexos I, III, IV, V e varios RNAs transportadores. Nao encontramos correlacao entre gravidade clinica, achados morfologicos e tamanho, localizacao e quantidade do DNAmit mutante, sugerindo que ha outros fatores influenciando o fenotipo da doencaBV UNIFESP: Teses e dissertaçõe