Morphological alterations in human skeletal muscle cells exposed to di-(2-Ethylhexyl) Phthalate (dehp) in primary cell culture conditions / Alteraciones morfológicas en células musculares esqueléticas humanas expuestas a Ftalato de di-(2-etilhexilo) (dehp) en condiciones de cultivo celular primario

SUMMARY: Di-(2-ethylhexyl) phthalate (DEHP) is among the most common plasticizer additives that humans are in contact with daily. DEHP can be released from plastic and enter the human body, whereby it is metabolized and transformed into oxidative hydrophilic molecules. Clinical follow-ups in patients exposed to this phthalate and investigations in cultures of several cell types have provided information on its effects. For example, it is associated with inhibition of diploid human cell development and morphological changes in cultured germ cells. Although skeletal muscle represents around 50 % of the human body mass, knowledge about the effects of DEHP on this tissue is poor. Cultured skeletal muscle cells were exposed to DEHP (1 mM) for 13 days with the aim of exploring and evaluating some of the potential morphological effects. Three culture development parameters and nine cell characteristics were monitored during the bioassay. At 13 days, growth area, cell viability, and concentration of total proteins were lower in DEHP exposed than in control cells. Cell width and area, as well as the diameter of the nucleus and nucleolus, were greater in exposed cells than in control cells. These are interpreted as signs of cytotoxicity and suggest potential adverse effects on the development of skeletal muscle cells from DEHP exposure, as reported for other cell types.

RESUMEN: Diariamente los seres humanos tenemos contacto con aditivos plastificantes, el di-(2-etilhexil) ftalato (DEHP) se encuentra entre los más comunes. El DEHP puede liberarse del plástico e ingresar al cuerpo humano, donde es metabolizado y transformando en moléculas hidrofílicas oxidativas. Seguimientos en pacientes expuestos a este ftalato e investigaciones en cultivos de varios tipos celulares han aportado información sobre sus efectos. El DEHP es asociado con la inhibición del desarrollo de células humanas diploides y cambios morfológicos en células germinales en cultivo. Sin embargo, aún es poco lo que se sabe sobre los efectos en el músculo esquelético, a pesar de que este tejido representa alrededor del 50 % de la masa corporal del humano. Para explorar y evaluar algunos efectos morfológicos en células de músculo esquelético, cultivos primarios fueron expuestos a DEHP (1 mM) durante 13 días. Se dio seguimiento a tres parámetros de desarrollo del cultivo y nueve características celulares. Al término de 13 días de exposición, los valores del área de crecimiento, viabilidad celular y concentración de proteínas totales fueron inferiores con respecto a los cultivos control. Se observaron cambios morfométricos en las células expuestas. Particularmente, el ancho y área celular, así como los diámetros del núcleo y nucleolos, fueron mayores a los registros en las células control. Estos resultados se interpretan como signos de citotoxicidad y sugieren efectos potencialmente adversos en el desarrollo de las células del músculo esquelético ante una exposición al DEHP, como se ha registrado para otros tipos celulares.

Morphometric characteristics of human skeletal muscle cells in primary culture / Características morfométricas de células musculares esqueléticas humanas en cultivo primario

SUMMARY: The study of cell morphology has contributed to the innovation of clinical techniques and biomedical research. Primary cell culture techniques are well standardized; however, knowledge about morphometric parameters under cell culture conditions is scarce. Variations in morphology can affect cell physiology and responses. The aim of this study was to use morphometric tools to describe the growth and development of skeletal muscle cells under standard cell culture conditions. A photographic database was generated, and morphometric data was obtained for nine cell characteristics (n = 559 cells). Four muscular cell shapes (spherical, irregular outline, triangular and spindle/fusiform) were characterized with wide ranges in variation. The maximum cell length (110-262 µm), width (35-66 µm), area (2,642 - 9,480 µm2), projection lengths (45 - 127 µm), and nucleus diameter (28 ± 11 µm) were obtained by day 23 of culture. A single centrally positioned nucleus was observed in each cell; nucleoli diameter (5 ± 2 µm) and number (1 - 5) varied. In general, cyclic changes in cell sizes were identified during culture, whereas cell length, width, and area increased in spurts. These results suggest that morphometric parameters can be used to monitor skeletal muscle cell development under standard culture conditions.

RESUMEN: A partir de células madre musculares, surgen los mioblastos que se dividen y fusionan entre sí para formar a los miocitos. Estas células ya diferenciadas son precursoras de miocitos que maduran en fibras musculares y posteriormente forman los músculos. La implementación de cultivos celulares de mioblastos ha permitido obtener conocimiento detallado del tejido muscular. Particularmente, algunas de las aportaciones morfológicas fueron el punto de partida de técnicas clínicas, terapias o investigaciones biomédicas. Sin embargo, los estudios morfométricos en condiciones de cultivo celular son escasos. Por lo cual, realizamos seguimientos fotográficos a cultivos desarrollados bajo condiciones estándar, registramos datos para nueve características celulares y aplicamos técnicas morfométricas para analizar estas células (n = 559). Se caracterizaron cuatro formas celulares adoptadas por los mioblastos (esférica, irregular, triangular y huso) y se registraron intervalos amplios de variación en los caracteres. Hacia el día 23 de cultivo se presentaron los valores máximos en la longitud (110-262 µm), el ancho (35-66 µm) y el área celular (2,642-9,480 µm2), así como en el tamaño máximo de las proyecciones celulares (45-127 µm) y el diámetro del núcleo (28±11 µm). El núcleo se observó como único y en posición central; los nucleolos variaron poco en diámetro (5±2 µm), aunque no en número (1 a 5). En términos generales, se identificaron cambios cíclicos en la talla de las células durante los cultivos, esto es, períodos intercalados de incremento y decremento en el largo, ancho y área celular. Debido a que estas características reflejaron los cambios generales sufridos por los mioblastos durante el cultivo, se proponen para monitorear sus etapas de desarrollo en cultivo.

CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

Background: Myostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn−/+ KO cells, and wondered whether the mitochondria biogenesis are affected. Results: In this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels. Conclusion: These findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.

Fine needle aspiration cytology in feline skeletal muscle as a diagnostic tool for extramedullary plasmacytoma / Citologia aspirativa por agulha fina no músculo esquelético felino como ferramenta diagnóstica para o plasmocitoma extramedular

Extramedullary noncutaneous plasmacytoma (ENP) is a myeloproliferative disorder of plasma cells that rarely affects cats. This paper describes an ENP case revealed by fine needle aspiration cytology (FNAC) of the mass in the skeletal muscle of an 8-month-old, male, mixed breed cat, which had a nodule in the left hind limb. The rapid immunoassay test confirmed the presence of feline leukemia virus (FeLV). The animal necropsy macroscopically showed the nodule came from the semimembranosus muscle. Histopathological examination ratified the cytological findings. Thus, this paper alerts to the existence of plasmacytoma located in the skeletal muscle of feline species. FNAC is a quick and efficient method for diagnosis of ENP.(AU)

O plasmocitoma extramedular (PEM) não cutâneo é um distúrbio mieloproliferativo de plasmócitos que raramente acomete felinos. Este trabalho descreve um caso de PEM no músculo esquelético de um gato, macho, sem raça definida, de oito meses de idade, que apresentava um aumento de volume no membro pélvico esquerdo. A citologia aspirativa por agulha fina (CAAF) da massa revelou tratar-se de PEM. O teste imunoenzimático rápido confirmou a presença do vírus da leucemia felina (FeLV). Na necropsia do animal, macroscopicamente, percebeu-se que o nódulo era originário do músculo semimembranoso. O exame histopatológico ratificou os achados citológicos. Desta forma, alerta-se para a existência de plasmocitoma com localização em músculo esquelético na espécie felina, sendo a CAAF um método alternativo rápido e eficaz para o seu diagnóstico.(AU)

Regulation of muscle plasticity and trophism by fatty acids: A short review / Regulação da plasticidade e do trofismo muscular pelos ácidos graxos: uma breve revisão

Summary The skeletal muscle tissue has a remarkable ability to alter its plastic structural and functional properties after a harmful stimulus, regulating the expression of proteins in complex events such as muscle regeneration. In this context, considering that potential therapeutic agents have been widely studied, nutritional strategies have been investigated in order to improve the regenerative capacity of skeletal muscle. There is evidence of the modulatory action of fatty acids, such that oleic and linoleic acids, that are abundant in Western diets, on muscle function and trophism. Thus, fatty acids appear to be potential candidates to promote or impair the recovery of muscle mass and function during regeneration, since they modulate intracellular pathways that regulate myogenesis. This study is the first to describe and discuss the effect of fatty acids on muscle plasticity and trophism, with emphasis on skeletal muscle regeneration and in vitro differentiation of muscle cells.

Resumo O tecido muscular esquelético possui a notável capacidade plástica de alterar suas propriedades estruturais e funcionais após um estímulo lesivo, regulando a expressão de proteínas durante eventos complexos como a regeneração muscular. Nesse contexto, considerando que possíveis agentes terapêuticos vêm sendo amplamente estudados, estratégias nutricionais têm sido investigadas na perspectiva de melhorar a capacidade regenerativa do músculo esquelético. Há evidências da ação modulatória dos ácidos graxos, como os ácidos oleico e linoleico, que são abundantes nas dietas ocidentais, sobre a função muscular e o trofismo. Nesse sentido, os ácidos graxos parecem ser potenciais candidatos para promover ou prejudicar a recuperação da massa e a função muscular durante a regeneração, uma vez que modulam vias intracelulares reguladoras da miogênese. Este trabalho é o primeiro a descrever e discutir o efeito dos ácidos graxos sobre a plasticidade e o trofismo muscular, com ênfase na regeneração do músculo esquelético e na diferenciação de células musculares in vitro.

Prevalência de autorrelato de diagnóstico médico de doença renal crônica no Brasil: Pesquisa Nacional de Saúde, 2013 / Prevalence of self-reported chronic kidney disease in Brazil: National Health Survey of 2013

RESUMO: Objetivo: Descrever o perfil dos adultos que referiram diagnóstico médico de doença renal crônica (DRC), segundo variáveis selecionadas. Métodos: Estudo transversal em que foram incluídos indivíduos entrevistados pela Pesquisa Nacional de Saúde de 2013, estudo de base populacional e domiciliar realizado no Brasil, representativo da zona rural e urbana. Foram avaliados 60.202 indivíduos com idade ≥ 18 anos que referiram diagnóstico médico de insuficiência renal crônica ou doença renal. Foi realizada estatística descritiva, incluindo cálculos de prevalências e respectivos intervalos de confiança de 95% (IC95%). Resultados: A prevalência de DRC foi de 1,4% (IC95% 1,3 - 1,6), semelhantes entre os sexos; masculino: 1,4% (IC95% 1,1 - 1,6) e feminino 1,5% ((IC95% 1,3 - 1,7). A região Sul apresentou a maior frequência desse indicador (2,1%; IC95% 1,6 - 2,7). A prevalência de tratamento dialítico dentre as pessoas com diagnóstico médico autorreferido de DRC foi de 7,4% (IC95% 4,4 - 10,3), sendo maior no sexo masculino (12,4%; IC95% 6,5 - 18,3) e não houve diferença entre as faixas etárias e os níveis de escolaridade. DRC foi referida por 8,9% (IC95% 3,5 - 14,3) dos pardos, sem diferença entre as raças/cor da pele. Conclusão: Esses resultados revelam os diversos aspectos da DRC no país. Observou-se que a distribuição foi desigual, onerando principalmente os de menor escolaridade, o que demanda maior investimento em programas de saúde para o enfrentamento dessa enfermidade. Dessa forma, esses dados permitem direcionar o planejamento de políticas públicas voltadas à prevenção dessa doença e à promoção da saúde.

ABSTRACT: Objective: To describe the profile of adults who reported medical diagnosis of chronic kidney disease (CKD), according to selected variables. Methods: In a cross-sectional study with individuals included in the National Health Survey of 2013, a household population-based study was conducted in rural and urban areas of Brazil. A total of 60,202 individuals aged ≥18 years who self-reported a medical diagnosis of chronic renal failure or kidney disease were evaluated. Descriptive statistics, including calculations of prevalence and 95% confidence intervals (95%CI), were calculated. Results: The prevalence of CKD was 1.4% (95%CI 1.3 - 1.6). It was similar between sexes: male, 1.4% (95%CI 1.1 - 1.6); and female, 1.5% ((95%CI 1.3 - 1.7). southern Brazil showed the highest frequency of this indicator (2.1%; 95%CI 1.6 - 2.7). The prevalence of dialysis among people with medical diagnosis of end stage renal disease was 7.4% (95%CI 4.4 - 10.3), being greater in males (12.4%; 95%CI 6.5 - 18.3). There was no difference between the age groups and schooling levels. CKD was referenced by 8.9% (95%CI 3.5 - 14.3) of the individuals with brown skin, with no difference among races/skin color. Conclusion: These results reveal various aspects of CKD in Brazil. The distribution of CKD was unequal, burdening especially individuals with poor education, demanding greater investments in health programs for the confrontation of CKD. Thus, these data allow the planning of public policies aimed at the prevention of this disease and health promotion.

Expression levels of sarcolemmal membrane repair proteins following prolonged exercise training in mice.

Membrane repair is a conserved cellular process, where intracellular vesicles translocate to sites of plasma membrane injury to actively reseal membrane disruptions. Such membrane disruptions commonly occur in the course of normal physiology, particularly in skeletal muscles due to repeated contraction producing small tears in the sarcolemmal membrane. Here, we investigated whether prolonged exercise could produce adaptive changes in expression levels of proteins associated with the membrane repair process, including mitsugumin 53/tripartite motif-containing protein 72 (MG53/TRIM72), dysferlin and caveolin-3 (cav3). Mice were exercised using a treadmill running protocol and protein levels were measured by immunoblotting. The specificity of the antibodies used was established by immunoblot testing of various tissue lysates from both mice and rats. We found that MG53/TRIM72 immunostaining on isolated mouse skeletal muscle fibers showed protein localization at sites of membrane disruption created by the isolation of these muscle fibers. However, no significant changes in the expression levels of the tested membrane repair proteins were observed following prolonged treadmill running for eight weeks (30 to 80 min/day). These findings suggest that any compensation occurring in the membrane repair process in skeletal muscle following prolonged exercise does not affect the expression levels of these three key membrane repair proteins.

Analysis of osmotic stress induced Ca2+ spark termination in mammalian skeletal muscle.

Ca2+ sparks represent synchronous opening of the ryanodine receptor (RyR) Ca2+ release channels located at the sarcoplasmic reticulum (SR) membrane. Whereas a quantal nature of Ca2+ sparks has been defined in cardiac muscle, the regulation of Ca2+ sparks in skeletal muscle has not been well-studied. Osmotic-stress applied to an intact skeletal muscle fiber can produce brief Ca2+ sparks and prolonged Ca2+ burst events. Here, we show that termination of Ca2+ bursts occurs in a step wise and quantal manner. Ca2+ burst events display kinetic features that are consistent with the involvement of both stochastic attrition and coordinated closure of RyR channels in the termination of SR Ca2+ release. Elemental unitary transition steps could be defined with a mean DF/F0 of ~0.28, corresponding to the gating of 1-2 RyR channels. Moreover, the amplitude of the elemental transition steps declines at the later stage of the burst event. In tandem Ca2+ burst events where two Ca2+ bursts occur at the same position within a fiber in rapid succession, the trailing event is consistently of lower amplitude than the initial event. These two complementary results suggest that SR Ca2+ release may be associated with local depletion of SR Ca2+ stores in mammalian skeletal muscle.

Correlation between Innervation of Skeletal Muscles and Myoblast Stem Cells / Correlación entre la Inervación de los Músculos Esqueléticos y las Células Precursoras Mioblásticas

In order to explore the change rule of myoblast stem cells (satellite cells, SCs) in the denervated and re-innervated muscle and to investigate the cellular mechanism of the morphological and functional changes of the muscle, denervated muscle atrophy and nerve regeneration models were established in one-month-old rats. Postoperative indexes such as muscle wet weight, cell section areas, content of collagen fibers and DNA, electrophysiology, numbers of SCs in the triceps muscle of calf were dynamically tested. After denervation, the muscle wet weight and cell area reduced rapidly, and the collagen fiber content increased slowly. The number of SCs increased at first, and then declined suddenly two months later. From 4 to 5 weeks after re-neuralization, muscle action potentials could be evoked, but the best innervation effect was found in the groups, which received re-neuralization at 2 months and 3 months after denervation. Denervation causes a progressive progress of muscle atrophy. SCs proliferate within 3 months after denervation, and then atrophy becomes irreversible from 4 months. At 4 or 5 weeks after re-neuralization, muscle action potentials can be evoked. Re-neuralization at 2 months and 3 months after denervation can achieve a good effect on the functional recovery of the atrophic muscle.

Con el fin de explorar la regla de cambio de las células precursoras mioblásticas (células satélite, CSs) en el músculo denervado y re-inervado e investigar el mecanismo celular de los cambios morfológicos y funcionales del músculo, se establecieron, en ratas de un mes de edad, modelos de atrofia del músculo denervado y regeneración del nervio. Fueron examinados de manera dinámica índices postoperatorios tales como, el peso húmedo del músculo, áreas celulares de la sección, contenido de fibras de colágeno y ADN, electrofisiología, número de CSs en el músculo tríceps de las crías. Luego de la denervación, el peso del músculo húmedo y el área celular se redujeron rápidamente, mientras que el contenido de fibras colágenas aumentó lentamente. El número de CSs aumentó al inicio, pero más tarde, a los dos meses, disminuyó repentinamente. Entre las 4 a 5 semanas después de la reneuralización, los potenciales de acción muscular pudieron ser evocados, pero el mejor efecto de inervación se encontró en los grupos que recibieron reneuralización a los 2 y 3 meses después de la denervación. La denervación causó un avance progresivo de la atrofia muscular. Las CSs proliferaron dentro de los primeros 3 meses post-denervación, y luego de los 4 meses la atrofia fue irreversible. A las 4 o 5 semanas después de la reneuralizacón, los potenciales de acción muscular pueden ser evocados. La reneuralización a los 2 y 3 meses después de la denervación puede lograr un buen efecto en la recuperación funcional del músculo atrófico.

O papel da citologia no diagnóstico das neoplasias músculo-esqueléticas: revisão sistemática / The role of cytology in the diagnosis of musculoskeletal neoplasms: systematic review

Os autores revisaram sistematicamente a literatura da última década sobre o papel ocupado pela citologia na avaliação das neoplasias músculo-esqueléticas e sua precisão diagnóstica. Foi realizada uma consulta nas bases de dados PubMed, MEDLINE, LILACS e SciELO, que utilizassem a citologia no diagnóstico das lesões músculo-esqueléticas . Foram utilizados limites para os idiomas inglês, espanhol e português. e artigos publicados a partir de 2000. Foram resgatados 757 artigos, dos quais 24 foram selecionados com a aplicação dos critérios de inclusão e exclusão. Concluiu-se que apesar de promissora na avaliação das lesões músculo-esqueléticas, a citologia obtida por punção por agulha fina é menos precisa e confiável do que a avaliação histológica na avaliação dessas lesões.

The authors systematically reviewed the literature of the last decade on the role of cytology in theevaluation of musculoskeletal neoplasms, and its diagnostic accuracy. A search was carried out on the databases PubMed, MEDLINE, LILACS and SciELO, selecting articles in which cytology was used in the diagnosis of musculoskeletal neoplasms. Limits(Boolean operators?) were used for English, Spanish and Portuguese, and only articles published since2000 were selected. 757 articles were retrieved, 24 of which were selected based on criteria of inclusion and exclusion. It was concluded that although promising in the assessment of musculoskeletal neoplasms, cytology obtained by fine needle aspiration is less accurate and reliable than histological evaluation of such lesions.

Histological description of the interaction between muscle fibers and connective tissue of the fascia of the human trapezius muscle / Descripción histológica de la interacción entre las fibras musculares y tejido conectivo de la fascia del músculo trapecio humano

The skeletal muscle fascia corresponds to a condensation of connective tissue. Fascias are highly innervated and sensitive, and can cover non-expandable structures as well as musculature. It is suggested that fascias have a pivotal role in functions such as postural regulation, peripheral motor coordination and proprioception. Also, the presence of inflammation and microcalcification in fascia of patients with localized muscle pain has been described, suggesting a pathogenic role in pain. The aim was to describe the histological structure of the external deep fascia of the trapezius muscle, with emphasis on the content and arrangement of muscle fibers, type I collagen, and adipose tissue. Sample material was obtained from a male cadaver (60-70 years old), by dissection of the posterior cervical region of the superficial fascia of the trapezius muscle and fixed in buffered formalin. Samples were processed by routine histological techniques and embedded in paraffin, obtaining 5 µm-thick sections that were stained according to the van Gieson technique. The trapezius fascia is composed of type I collagen, organized into high-density collagen bundles and oriented in different directions, and by adipocytes disposed in longitudinal groups on the main axis of the fascia. Muscle fibers are organized into bundles that are inserted laterally on the thickness of the fascia. It is possible that lateral transmission of tensional forces between the fibers might be present.

La fascia del músculo esquelético corresponde a una condensación de tejido conectivo. Las fascias están inervadas y son sensibles y pueden cubrir estructuras no distensibles, así como las fibras musculares esqueléticas. Tienen un rol importante en la regulación de la postura, la coordinación motora periférica y la propiocepción. Además, se ha descrito la presencia de inflamación y microcalcificaciones en la fascia de los pacientes con dolor muscular localizado, lo que sugiere un rol patogénico en el dolor. El objetivo del trabajo fue describir la estructura histológica de la fascia externa profunda del músculo trapecio, con énfasis en el contenido y la disposición de las fibras musculares, colágeno tipo I y el tejido adiposo. Material y métodos: El material de la muestra fue obtenido de un cadáver de sexo masculino (60-70 años), por la disección de la región cervical posterior de la fascia superficial del músculo trapecio e inmediatamente fijado en formalina tamponada (pH 7,2) durante 48 horas. La muestra fue procesada por técnicas histológicas e impregnada en parafina (punto de fusión 56-58 C). Secciones de 5 µm de espesor fueron montadas en láminas silanizada para el desarrollo del protocolo de la técnica de van Gieson. Resultados y discusión: Se observa que la fascia del trapecio está compuesta por tejido conectivo denso irregular con abundante colágeno tipo I, organizado en paquetes grandes como verdaderos haces de colágeno de alta densidad orientada en diferentes direcciones; y por adipocitos dispuestos en grupos longitudinales en el eje principal de la fascia. Las fibras musculares estriadas están organizadas en paquetes que se insertan lateralmente en el espesor de la fascia. Es posible que la transmisión lateral de la tensión entre las fibras esté presente.

Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Molecular & genetic factors contributing to insulin resistance in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of unknown etiology. Insulin resistance is very common and plays a central pathogenic role in PCOS. During last decade several studies have been conducted to understand the mechanisms contributing to the state of insulin resistance and insulin-induced hyperandrogenemia in PCOS. Insulin signaling pathways have been dissected in different insulin responsive tissues such as skeletal muscles, adipose tissues, fibroblasts as well as ovaries to elucidate the mechanism. These studies suggest a post receptor signaling defect where metabolic action of insulin is affected but not the steroidogenic and mitogenic actions. Despite advancement in these studies gaps exist in our understanding of the mechanism of insulin resistance as well as insulin-induced steroidogenesis in PCOS. The syndrome is now considered as a complex multigenic disorder. Efforts are ongoing to dissect the variants of genes from multiple logical pathways which are involved in pathophysiology of the syndrome. But still today no gene has been emerged as universally accepted susceptibility gene for PCOS. This review briefly describes the lacunae along with the current status of molecular events underlying insulin resistance and the contribution of insulin signaling pathway genes in pathogenesis of PCOS along with future researchable areas.

Histological study of fresh versus frozen semitendinous muscle tendon allografts

OBJECTIVE: The purpose of this study was to histologically analyze allografts from cadaveric semitendinous muscle after cryopreservation at -80°C in comparison to a control group kept at only -4°C to test the hypothesis that the histological characteristics of the tissue are maintained when the tendons are kept at lower temperatures. METHODS: In a tissue bank, 10 semitendinous tendons from 10 cadavers were frozen at -80ºC as a storage method for tissue preservation. They were kept frozen for 40 days, and then a histological study was carried out. Another 10 tendon samples were analyzed while still "fresh". RESULTS: There was no histological difference between the fresh and frozen samples in relation to seven variables. CONCLUSIONS: Semitendinous muscle tendon allografts can be submitted to cryopreservation at -80ºC without suffering histological modifications.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3 percent of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Cytotoxicity and morphological analysis of cell death induced by Bothrops venoms from the northeast of Argentina

Bothrops snake venoms have been proved toxic to a variety of cell types, in both in vivo and in vitro models. Studies on the pharmacological actions of Bothrops venoms from Argentina are relatively secarce and the direct action of the crude venoms has not been assessed using cell culture models. In this work, we investigated the cytotoxicity of crude venoms from B. alternatus and B. diporus in a skeletal muscle (C2C12) cell line, which is commonly used as a model for studying the myotoxic action of snake venom. Both venoms (1.25-50 miug/mL) induced an early and significant decrease in cell viability. The cytotoxic concentration 50 (CC50), determined three hours after exposure, revealed that B. diporus venom was significantly more cytotoxic (CC50: 2 miug/mL) than B. aftematus (CC50: 5.8 miug/mL). To investigate the cell death mechanism involved, myoblast cells were examined by phase contrast microscopy and after acridine orange and ethidium bromide fluorescence staining, respectively. Our data clearly demonstrated that an apoptotic mediated this cell line destruction. The current study aimed to provide new information on the citotoxicity meohanisms of Argentine Bothrops snake venoms on a skeletal muscle cell line

Disposition of the striated urethral sphincter and its relation to the prostate in human fetuses

OBJECTIVE: To describe the arrangement of the muscle fibers of the striated urethral sphincter and its relationship with the prostate during the fetal period in humans. MATERIALS AND METHODS: We analyzed 17 prostates from well preserved fresh human fetuses ranging in age from 10 to 31 weeks postconception (WPC). Transversal sections were obtained and stained with Gomori's trichrome and immunolabeled with anti alpha-actin antibody. RESULTS: We found that the urethral striated sphincter (rabdosphincter) is located on the periphery of the smooth muscle and there was no merge between striated and smooth muscle fibers in any fetal period. In the prostate apex, the striated sphincter shows a circular arrangement and covers completely the urethra externally, whereas adjacent to verumontanum, it looks like a "horseshoe" and covers only the anterior and lateral surfaces of the urethra. Near the bladder neck, in fetuses younger than 20 WPC, we have found striated muscle fibers only at the anterior surface of the prostate, while in fetuses older than 20 WPC, the striated muscle covers the anterior and lateral surfaces of the prostate. CONCLUSIONS: The urethral sphincter muscle covers the anterior and lateral surfaces of the urethra in all fetuses older than 20 WPC, close to the bladder neck and at the distal prostate. In the region of the prostate apex, the urethral sphincter covers completely the urethra circularly. The knowledge of the normal anatomy of the urethral sphincter in fetuses could be important to understand its alterations in congenital anomalies involving the base of the bladder, the bladder neck and the proximal urethra.

Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells

Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.

Implante cardíaco de mioblastos. Resultado en el seguimiento a tres años / Myoblast trnsplantation for cardiac repair: a three-year follow up

Este trabajo se analiza como una continuación de una de las primeras experiencias publicadas en el mundo sobre implante clínico de células con mioblastos, cuyos resultados fueron editados en este mismo órgano de difusión. Si tomamos en cuenta los segmentos comprometidos y los dividimos en infarto transmural, infarto no transmural, isquémicos y normales, hallamos que sobre 68 segmentos pasibles de estudio en los 4 enfermos sobrevivientes a los 33 ± 6,05 meses hubo un claro retroceso de los segmentos con infarto transmural y un incremento en los segmentos no transmurales e isquémicos. En los segmentos con compromiso transmural, éstos retrocedieron de 15 a 3, lo cual representa una reducción del 80 por ciento (p = 0,0005). El análisis de los segmentos no transmurales debe ser exhaustivo. Si bien globalmente aumentaron de 7 a 10, los segmentos no transmurales registrados originariamente en el preoperatorio descendieron de 7 a 2, un 72 por ciento. El aumento global de estos segmentos no transmurales puede explicarse por los segmentos incorporados tanto por el avance de la enfermedad como a expensas de los transmurales, en claro retroceso del tejido fibrótico.

Calcium handling in a testosterone responsive skeletal muscle of the MDX mouse

Muscle necrosis in Duchenne muscle dystrophy (DMD) and in the mdx mouse has been related to abnormal calcium homeostasis associated with the lack of dystrophin. We have previously shown that the testosterone-dependent levator ani (LA) muscle of the mdx mouse develops a mild muscle wasting and fiber degeneration compared to the less hormone sensitive diaphragm (DIA) muscle, suggesting a protective effect of androgens. This study assessed the calcium handling mechanisms and cytosolic calcium concentration ([Ca2+]i) in LA muscles of mdx mice at critical stages of muscle disease. Muscle contractures induced by caffeine and 4-chloro-m-cresol (4-CmC), two activators of ryanodine channels, were recorded in LA and DIA muscles of prepubertal (1 month-old), adult (4 month-old) and aged (18 month-old) wild-type (wt) and mdx mice. [Ca2+]i was estimated with the fura-2 fluorescent dye in enzymatically dissociated LA muscle fibers of the same wt and mdx groups. Tetanus tension (TT) in the LA increased proportionately to the muscle weight (4 to 5-fold), but specific TT (TT/mg) did not differ among age-matched wt and mdx groups. Muscle contractures induced by caffeine (3-100 mM) or 4-CmC (0.1-5.0 mM) in the LA were greater in prepubertal than in adult and aged mice, but they did not differ among age-matched wt and mdx groups. The resting [Ca2+]i in mdx LA muscle fibers was not significantly affected at any age. Comparatively, dystrophic DIA presented reduced muscle strength in adult (40%) and aged (45%) mice, whereas the muscle responses to caffeine increased with age (63 to 82%), indicating changes in the Ca2+ handling mechanisms. The results indicated that muscle strength and calcium homeostasis in dystrophic LA muscle fibers were not significantly altered, confirming previous evidence of androgens’ beneficial effects on hormone-sensitive skeletal muscles.