Este trabalho foi apoiado por um Serviço de Saúde Pública Grant (HL086513) de NHLBI a PEO, e uma bolsa de pesquisa abrangente do Instituto de Medicina Regenerativa da Califórnia (RC1-00104), um Serviço de Saúde Pública Grant (HL085377) de NHLBI, e um presente da Fundação Pollin para HSB SC foi apoiado por uma California Institute for Bridges Medicina Regenerativa de Stem Prêmio Célula de Investigação (TB1-01194) para San Francisco State University.

O protocolo é realizada com a aprovação do Animal Care UCSF Institucional e Comitê de Uso (IACUC). 1. Dissecção da Longus do rato Digitorum Extensor (EDL) Músculo <ol><li> Executar todos os procedimentos animal, em conformidade com as diretrizes institucionais. </li><li> Eutanásia de animais com 200 mg / kg deslocamento pentobarbital / cervical intraperitoneal apenas antes da colheita do músculo 10. A dissecção deve ser bem praticada para que o músculo pode ser colhido e montada no transdutor de tensão dentro de 15 minutos de eutanásia. </li><li> Organizar supina carcaça na bandeja de dissecação e perna pino para bandeja. </li><li> Sob o microscópio de dissecação, aberta pele, fáscia cuidadosamente aberto (Fig.1a), e casca tibial do tornozelo para cima para expor EDL (Fig.1b). Use gotas de solução de Ringer com lactato para manter o músculo úmido e buffer durante a colheita. </li><li> Remover EDL, preservando como tendão tanto quanto possível em cada extremidade, e colocado em uma placa de Petri contendo solução de Ringer com lactato. Laço de sutura para cada um dos tendões do músculo (Figura 1C). É essencial que as fibras musculares não ser tocado ou perturbado durante a dissecção. </li></ol> 2. Montagem da EDL do rato na Faixa de Myograph Muscle <ol><li> Para esses estudos, um banho de tecido é necessário que prende o músculo ao mesmo tempo permitindo que ele se banhar em soro fisiológico à temperatura constante com oxigenação contínua. O banho é acoplado a um transdutor de força para a medição da tensão muscular. Nós empregamos um sistema integrado de banho myograph muscular tira do dinamarquês Myo Tecnologia (DMT Modelo 820MS) para esta finalidade. Além disso, um estimulador de pulsos elétricos quadrados (Modelo Grass S48) e dados da plataforma de aquisição (ADInstruments PowerLab Sistema de aquisição de dados e software LabChart) são necessários para obter, registrar e analisar mio-mecânica respostas, respectivamente. A 820MS DMT tem eletrodos de platina integrado a tampa do compartimento que estão posicionados em ambos os lados do músculo, na porção média da faixa de músculo. Myographs outras podem necessitar de cuidados específicos para a colocação do eletrodo. </li><li> Preencha banho myograph com 5 mL de solução Krebs Henseleit 11. Quente a 25 ° C. Bolha O 2 / CO 2 (95% / 5%) através de banho por 15 minutos antes do uso. </li><li> Use suturas para estender EDL entre pinças de myograph e segura os tendões do músculo EDL (Fig.1D, E) entre as pinças. Tenha cuidado para não prender o próprio músculo. </li><li> Manter myograph banho a 25 ° C. </li></ol> 3. Myo-mecânico Análise A. tensão Twitch <ol><li> Definir o comprimento inicial no banho para que não haja flacidez muscular. </li><li> Determine estímulo máximo (duração 0,5 ms), ajustando a tensão, a fim de obter a tensão máxima contração muscular, em seguida, definir estímulo de 20% acima da máxima (para alcançar estímulo supramáxima). Em nossos estudos, o estímulo supramáximo é geralmente obtida em uma saída de 40 volts. </li><li> Verifique a saída do estimulador usando um osciloscópio. </li><li> Determinar o comprimento ideal gradualmente alongamento do músculo até que não haja aumento de tensões twitch. </li><li> Permitir muscular para equilibrar por 3 min. </li><li> Oferecer estímulo quadrados supramáximo (0,5 ms) no comprimento ideal usando grama S44 estimulador eletrônico e de saída de gravação. </li><li> Record: curva de tensão twitch (P t vs tempo; Fig.2A). </li></ol> B. Tétano tensão <ol><li> Permitir muscular para descansar por 3 minutos. </li><li> Aplicar um trem de estímulos supramáximos para 300msec a 150 Hz no comprimento ideal usando grama S44 estimulador eletrônico e de saída de gravação. </li><li> Record: tétano curva tensão (P o tempo vs; Fig.2B). </li></ol> C. Força-frequência <ol><li> Permitir muscular para descansar por 3 minutos. </li><li> Força-freqüência: aplicar trens de estímulos supramáximos aos 30, 60, 100, 140 e 160 Hz, com 3 minutos de descanso entre cada estímulo (Fig. 3). Alternativamente, os trens podem ser aplicadas 15, 25, 35, 45, 55, 65, 75, 100, 140 e 160 Hz para uma melhor resolução em freqüências mais baixas, onde a força muda substancialmente. </li><li> Parcela: relação força-frequência (% máxima força freqüência de estimulação vs). </li></ol> D. Fadiga <ol><li> Aplicar trem de tetani curto: 60 Hz para 300 ms (ou frequência ajustada para produzir 50% do pico de força), a cada 3 segundos por 10 minutos. Por 10 minutos, a força tetânica deve recusar-se a um nível de platô de ~ 15% do valor inicial (Fig.4). </li><li> Parcela: a fadiga de baixa freqüência (% vs tempo máximo vigor). </li></ol> E. coleta de dados adicionais no final do protocolo <ol><li> Antes de desmontar o músculo do myograph, defina o muscle no tamanho ideal, conforme determinado na etapa III.A.4 e medir seu diâmetro usando uma ocular no microscópio ou com pinças. Calcular área transversal (M 2). </li><li> Medida da massa muscular (mg), removendo suturas e pesando muscular. </li><li> Pesar do mouse para avaliar a massa corporal (gm). </li></ol> 4. Cálculos <ol><li> Relação músculo: massa corporal = massa muscular / massa corporal </li><li> Tensão Twitch, P t (mN) = tensão máxima gerada durante a contração </li><li> Tensão twitch específico (N / cm 2) = twitch tensão (MN) / área da seção transversal (M 2) x 10 5 N / M mN • 2 cm / 2 </li><li> Tempo para o pico de tensão (ms) = tempo do início da contração de tensão máxima </li><li> Tempo de relaxamento (ms) = tempo de pico de tensão para 50% do pico de tensão </li><li> Tetânica tensão, P O (MN) = tensão máxima gerada durante o tétano </li><li> Tensão tetânica específico (N / cm 2) = tetânica tensão (MN) / área da seção transversal (M 2) x 10 5 N / M mN • 2 cm / 2 </li><li> Taxa máxima de aumento de tétano (N / s) = taxa máxima de aumento da tensão durante a ascensão tensão no tétano, ou seja, a inclinação máxima da curva de tensão tetânica (ou, dP O / dt) </li><li> Metade de relaxamento de tensão tetânica (ms) = tempo da cessação da estimulação a 50% da tensão no término da estimulação </li><li> Twitch tensão-to-tetânica relação de tensão, P t / P O = máxima contração tensão tetânica tensão / máximo </li><li> Índice de fadiga = relação de tensão após dois minutos de fadiga de baixa freqüência para tensão isométrica máxima </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2582/2582fig1.jpg" alt="Figura 1" /> Figura 1. Dissecção do músculo EDL. A exposição, de hindlimb muscles.TA, tibial anterior. B, Exposição de EDL muscular (extensor longo dos dedos). C, Anexo de sutura para tendões EDL. D, banho Tension transdutor (vista do lado). E, EDL montados em banho (vista de cima). O músculo em forma incompleta imerso no buffer para fins ilustrativos, na prática, o músculo deve ser completamente imerso para evitar que sequem. <img src="/files/ftp_upload/2582/2582fig2.jpg" alt="Figura 2" /> Figura 2. Exemplo de curvas de tensão. Um exemplo, da curva de contração tensão ilustrando a tensão máxima contração (P t), tempo de contração (TC) e tempo de relaxamento (HRT). Bar, 1s. B, Exemplo de curva de tensão tetânica mostrando a tensão tetânica máxima (P o) e meia-relaxamento da tensão tetânica (HRTT). Bar, 1s. <img src="/files/ftp_upload/2582/2582fig3.jpg" alt="Figura 3" /> Figura 3. Exemplo de força de análise de frequências de relacionamento. A tensão, gerada por freqüências de estímulo incremental. B, Exemplo de trem de pulsos em 30MHz. Bar, 80ms. C, Exemplo de trem de pulsos em 140MHz. Bar, 80ms. D, Exemplo de força-freqüência curva de derivados de dados mostrados na A. A forma da curva força-frequência é característica da força muscular, e pode ser comparado entre os músculos de animais diferentes. <img src="/files/ftp_upload/2582/2582fig4.jpg" alt="Figura 4" /> Figura 4. Exemplo de análise de fadiga de baixa freqüência. A tensões, decrescente gerados ao longo do período de stimulation.Examples baixa freqüência de trens de pulso em pontos de tempo indicado (B, C, D) são apresentados abaixo. E, Exemplo de curva de baixa freqüência fadiga derivada de dados mostrado na A. A forma da curva de fadiga de baixa freqüência é característica de força muscular, e pode ser comparado entre os músculos de animais diferentes.

<ol>
	<li>Harcourt, L. J., Schertzer, J. D., Ryall, J. G., Lynch, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+dose+formoterol+administration+improves+muscle+function+in+dystrophic+mdx+mice+without+increasing+fatigue.">Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue.</a> Neuromuscul Disord. 17, 47-55 (2007).</li><li>Messina, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+overexpression+via+adeno-associated+virus+gene+transfer+promotes+skeletal+muscle+regeneration+and+enhances+muscle+function+in+mdx+mice.">VEGF overexpression via adeno-associated virus gene transfer promotes skeletal muscle regeneration and enhances muscle function in mdx mice.</a> FASEB J. 21, 3737-3746 (2007).</li><li>Danieli-Betto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+1-phosphate+protects+mouse+extensor+digitorum+longus+skeletal+muscle+during+fatigue.">Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue.</a> Am J Physiol Cell Physiol. 288, C1367-C1373 (2005).</li><li>MacIntosh, B. R., Willis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-frequency+relationship+and+potentiation+in+mammalian+skeletal+muscle.">Force-frequency relationship and potentiation in mammalian skeletal muscle.</a> J Appl Physiol. 88, 2088-2096 (2000).</li><li>Hayes, A., Williams, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+clenbuterol-treated+mdx+muscle+are+enhanced+by+low-intensity+swimming.">Contractile properties of clenbuterol-treated mdx muscle are enhanced by low-intensity swimming.</a> J Appl Physiol. 82, 435-439 (1997).</li><li>Coulton, G. R., Curtin, N. A., Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+skeletal+muscle+myopathy:+II.+Contractile+properties.">The mdx mouse skeletal muscle myopathy: II. Contractile properties.</a> Neuropathol Appl Neurobiol. 14, 299-314 (1988).</li><li>Danieli-Betto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+alpha-sarcoglycan+differently+affects+fast-+and+slow-twitch+skeletal+muscles.">Deficiency of alpha-sarcoglycan differently affects fast- and slow-twitch skeletal muscles.</a> Am J Physiol Regul Integr Comp Physiol. 289, R1328-R1337 (2005).</li><li>Chan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+for+speed:+contractile+properties+of+isolated+whole+EDL+muscle+from+an+alpha-actinin-3+knockout+mouse.">A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse.</a> Am J Physiol Cell Physiol. 295, C897-C904 (2008).</li><li>Personius, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grip+force,+EDL+contractile+properties,+and+voluntary+wheel+running+after+postdevelopmental+myostatin+depletion+in+mice.">Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice.</a> J Appl. , (2010).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Curr Protoc Immunol. Chap 1, (2006).</li><li>Beekley, M. D., Wetzel, P., Kubis, P., Gros, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscle+fibre+bundles+from+mice+deficient+in+carbonic+anhydrase+II.">Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II.</a> Pflugers Arch. 452, 453-463 (2006).</li></ol>

Não há conflitos de interesse declarados.

Descrevemos uma abordagem detalhada para avaliar mio-mecânicas no músculo explantado hindlimb do mouse. A EDL, enquanto mais difícil de dissecar por causa de sua posição posterior atrás do músculo tibial anterior, é mais fácil de avaliar que o tibial anterior devido à sua proeminente anexos tendinosa ao tornozelo e joelho. Esses tendões facilitar a montagem na myograph strip muscular. Em contraste, a mais facilmente acessada tibial anterior tem um anexo, ampla quase atendinous na articulação do joelho, tornando-se extremamente difícil tanto para dissecar, sem comprometer o músculo, e montar com segurança no myograph. Destacamos ainda que rapidamente a montagem do músculo em um banho de oxigenada em solução fisiológica e temperatura é essencial para preservar as propriedades mecânicas do músculo. Descobrimos que podemos repetir esta análise para até 30 minutos sem mudanças significativas na resposta do músculo sob essas condições. Finalmente, é essencial que as fibras musculares não ser tocado durante a dissecção e processos de montagem, pois isso pode ter efeitos adversos sobre a função muscular, e resultam em subestimação de mio-mecânico força. Seguindo estes procedimentos, esta análise fornece uma abordagem robusta quantitativa para avaliar os efeitos da modificação genética na função muscular 6,7,8,9, bem como a comparação entre intervenções terapêuticas em modelos do rato da doença muscular 1,2,3,5 .

Soluções e Mídia Solução de Ringer com lactato <ul><li> 100 mM NaCl </li><li> 30 mM CH3CH (OH) COONa (lactato de sódio) </li><li> 4 mM KCl </li><li> 1 mM CaCl 2 2H 2 O (cloreto de cálcio dihidratado) <ul><li> ajustar o pH a 6,75 </li></ul></li></ul> Krebs Henseleit solução <ul><li> 118 mM NaCl </li><li> 4,7 mM KCl </li><li> 1,25 mM CaCl 2 </li><li> 1,2 mM MgCl 2 </li><li> 1,2 mM KH 2 PO 4 </li><li> 25 mM NaHCO 3 </li><li> 11 mM de glicose <ul><li> ajustar o pH para 7,2-7,4 por equilibrar com O 2 / CO 2 (95% / 5%) de gás </li></ul></li></ul> Pentobarbital <ul><li> 5 mg / mL solução de trabalho em água estéril </li></ul>

myo-mechanical analysis of isolated skeletal muscle

Para avaliar os efeitos in vivo de intervenções terapêuticas para o tratamento da doença muscular 1,2,3, métodos quantitativos são necessários a medida da força e fadiga no músculo tratado. Descrevemos uma abordagem detalhada para avaliar mio-mecânicas em recém-explantado muscular hindlimb do mouse. Descrevemos a colheita atraumática do mouse músculo longus extensor dos dedos, a montagem do músculo em um músculo strip myograph (Modelo 820MS; Tecnologia Myo dinamarquês), ea medição da contração máxima e tensão tetânica, tempo de contração e relaxamento meia-hora, usando um quadrados de pulso estimulador (Modelo S48; Technologies Grass). Usando essas medidas, demonstramos o cálculo da contração muscular específica e tensão tetânica normalizada para a área muscular transversal, a contração-a-tetânica relação de tensão, a curva de relação força-freqüência ea curva de fadiga de baixa freqüência 4. Esta análise fornece um método para comparação quantitativa entre as intervenções terapêuticas em modelos do rato da doença muscular 1,2,3,5, bem como comparação dos efeitos de modificação genética em função muscular 6,7,8,9.

Watch this Scientific Journal Video about Myo-mechanical Analysis of Isolated Skeletal Muscle at JoVE.com

Myo-mechanical Analysis of Isolated Skeletal Muscle

Para avaliar a<em&gt; In vivo</em&gt; Efeitos de intervenções terapêuticas para doenças musculares, são necessários métodos para quantificar a geração de força e cansaço no músculo tratado. Detalhamos uma abordagem para avaliar mio-mecânicas no músculo de rato explantado hindlimb. Esta análise proporciona uma abordagem robusta para quantificação dos efeitos da modificação genética na função muscular, bem como a comparação de tratamentos em modelos do rato da doença muscular.

Myo-mecânico Análise do músculo esquelético isolado

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

myo-mechanical-analysis-of-isolated-skeletal-muscle

Research

JoVE Journal

Medicine

26.7K visualizações.  University of California San Francisco. Para avaliar a In vivo Efeitos de intervenções terapêuticas para doenças musculares, são necessários métodos para quantificar a geração de força e cansaço no músculo tratado. Detalhamos uma abordagem para avaliar mio-mecânicas no músculo de rato explantado hindlimb. Esta análise proporciona uma abordagem robusta para quantificação dos efeitos da modificação genética na função muscular, bem como a comparação de tratamentos em modelos ...

Vídeo: Myo-mechanical Analysis of Isolated Skeletal Muscle

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Dimorfismo Sexo músculo esquelético de Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Medicina

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

Secção do nervo tibial - um modelo padronizado para denervação induzida por atrofia muscular em ratos

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Procedimentos de biópsia de músculo esquelético humano, utilizando a técnica Bergström Modificado

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparação e Avaliação respirométrico de mitocôndrias isoladas de tecido muscular esquelético Obtido por meio de agulha de biópsia percutânea

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitativa Ressonância Magnética da doença do músculo esquelético

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Humano<em&gt; vasto lateral</em&gt; Biópsia de músculo esquelético Usando o Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Indução e Avaliação da Exertional lesão muscular esquelético em humanos

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

Semi automatizados de análise da morfologia do músculo esquelético de rato e composição de fibra-tipo

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Acoplamento de Neurovascular do músculo esquelético, capacidade oxidativa e função Microvascular com 'Balcão único' espectroscopia de infravermelho próximo

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force generating capacity. However, over 90% of all upper limb muscles and 85% of all lower limb muscles have optimal fascicle lengths longer than the field-of-view of common traditional ultrasound (T-US) probes. A newer, less frequently adopted method called extended field-of-view ultrasound (EFOV-US) can enable direct measurement of fascicles longer than the field-of-view of a single T-US image. This method, which automatically fits together a sequence of T-US images from a dynamic scan, has been demonstrated to be valid and reliable for obtaining muscle fascicle lengths in vivo. Despite the numerous skeletal muscles with long fascicles and the validity of the EFOV-US method for making measurements of such fascicles, few published studies have utilized this method. In this study, we demonstrate both how to implement the EFOV-US method to obtain high quality musculoskeletal images and how to quantify fascicle lengths from those images. We expect that this demonstration will encourage the use of the EFOV-US method to increase the pool of muscles, both in healthy and impaired populations, for which we have in vivo muscle fascicle length data.

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.