Ce travail a été soutenu par un Service de santé publique de subvention (HL086513) à partir du NHLBI à PEO, et une subvention de recherche global du California Institute for Regenerative Medicine (RC1-00104), un Service de santé publique de subvention (HL085377) à partir du NHLBI, et un cadeau de la Fondation Pollin à HSB SC a été soutenue par une California Institute for Regenerative Ponts souches Award Cell Research (TB1-01194) à San Francisco State University.

Le protocole est exécuté avec l&#39;approbation de l&#39;UCSF Soins des animaux et du Comité institutionnel d&#39;utilisation (IACUC). 1. Dissection de la souris Longus extenseur des orteils (EDL) Muscle <ol><li> Effectuez toutes les procédures animal conformément aux directives institutionnelles. </li><li> Euthanasier des animaux avec 200 mg / kg par voie intrapéritonéale du pentobarbital / col de l&#39;utérus juste avant la dislocation à 10 récoltes de muscle. La dissection doit être bien pratique pour que les muscles peuvent être récoltées et monté dans le transducteur de tension dans les 15 minutes de l&#39;euthanasie. </li><li> Disposer la carcasse en position couchée sur le plateau de dissection et de la jambe broches à plateau. </li><li> Sous jambier microscope à dissection, la peau ouverte, ouvrir avec précaution fascia (Fig.1A), et le zeste de la cheville vers le haut pour exposer les EDL (Fig. 1b). Utilisez des gouttes de solution de Ringer lactate pour garder le muscle humide et tamponnées pendant la récolte. </li><li> Retirer EDL, préservant le tendon autant que possible à chaque extrémité, et mis dans une boîte de Pétri contenant une solution de Ringer lactate. Tie suture pour chacun des tendons des muscles (Fig.1C). Il est essentiel que les fibres musculaires ne pas être touché ou dérangé pendant la dissection. </li></ol> 2. Le montage de l&#39;EDL la souris dans le myographe bande musculaire <ol><li> Pour ces études, un bain de tissu est nécessaire qui sécurise le muscle tout en lui permettant de se baigner dans une solution physiologique à température constante avec une oxygénation continue. Le bain est couplé avec un capteur de force pour la mesure de la tension musculaire. Nous employons un bain musculaire intégrés bande myographe du danois Myo Technologie (DMT modèle 820MS) à cet effet. En outre, un stimulateur électrique impulsion carrée (S48 Modèle Grass) et une plateforme d&#39;acquisition de données (ADInstruments PowerLab Système d&#39;acquisition de données et des logiciels LabChart) sont nécessaires pour susciter, enregistrer et analyser les réponses myo-mécaniques, respectivement. La DMT a 820MS électrodes de platine intégré dans le couvercle de la chambre qui sont positionnées de chaque côté du muscle, dans la partie médiane de la bande de muscle. Myographs autres peuvent nécessiter une attention particulière au placement des électrodes. </li><li> Remplissez bain myographe avec 5 ml de solution de Krebs Henseleit 11. Chaude à 25 ° C. Bubble O 2 / CO 2 (95% / 5%) dans le bain pendant 15 minutes avant utilisation. </li><li> Utilisez des sutures à étendre EDL entre les pinces du myographe et sécuriser les tendons du muscle EDL (Fig.1D, E) entre les pinces. Attention à ne pas serrer le muscle lui-même. </li><li> Maintenir bain myographe à 25 ° C. </li></ol> 3. Myo-mécanique Analyse Tensions Twitch A. <ol><li> Réglez la longueur initiale dans le bain afin qu&#39;il n&#39;y ait pas de relâchement musculaire. </li><li> Déterminer relance maximale (durée de 0,5 ms) en ajustant la tension afin d&#39;obtenir la tension maximale contraction, puis réglez de relance à 20% au-dessus maximal (pour atteindre relance supramaximale). Dans nos études, le stimulus supramaximale est habituellement réalisée sur une sortie de 40 volts. </li><li> Vérifiez la sortie du stimulateur à l&#39;aide d&#39;un oscilloscope. </li><li> Déterminer la longueur optimale en mettant graduellement étirer le muscle jusqu&#39;à ce qu&#39;il n&#39;y est aucune autre augmentation de la tension contraction. </li><li> Autoriser musculaire à s&#39;équilibrer pendant 3 min. </li><li> Livrer supramaximale relance carrés (0,5 ms) à longueur optimale à l&#39;aide d&#39;herbe S44 stimulateur électronique, et production record. </li><li> Record: la courbe de tension contraction (P t en fonction du temps; Fig.2a). </li></ol> B. Tétanos tensions <ol><li> Autoriser les muscles de se reposer pendant 3 minutes. </li><li> Appliquer un train de stimuli supramaximale pour 300msec à 150 Hz à longueur optimale à l&#39;aide d&#39;herbe S44 stimulateur électronique, et production record. </li><li> Record: la courbe de tension tétanos (P o vs temps; Fig.2b). </li></ol> C. force-fréquence <ol><li> Autoriser les muscles de se reposer pendant 3 minutes. </li><li> Force-fréquence: appliquer des trains de stimuli supramaximale à 30, 60, 100, 140 et 160 Hz avec 3 minutes de repos entre chaque stimulus (Fig.3). Alternativement, les trains peuvent être appliquées 15, 25, 35, 45, 55, 65, 75, 100, 140 et 160 Hz pour une meilleure résolution à des fréquences inférieures, où les changements force sensiblement. </li><li> Terrain: relation force-fréquence (fréquence maximale de stimulation% vs vigueur). </li></ol> D. fatigue <ol><li> Appliquer train de courte tetani: 60 Hz pendant 300 ms (ou fréquence ajustée pour produire 50% du pic de force), toutes les 3 secondes pendant 10 minutes. En 10 minutes, la force tétanique devrait diminuer à un niveau plateau de ~ 15% de la valeur initiale (Fig.4). </li><li> Terrain: la fatigue de basse fréquence (% temps maximal vs vigueur). </li></ol> Collecte de données supplémentaires E. à la fin du protocole de <ol><li> Avant de démonter le muscle de la myographe, réglez le muscLe moins longueur optimale déterminée à l&#39;étape III.A.4 et mesurer son diamètre en utilisant soit un oculaire sur le microscope ou avec étriers. Calculer section transversale (um 2). </li><li> Mesurer la masse musculaire (mg) par la suppression des sutures et pesant de muscle. </li><li> Peser la souris afin d&#39;évaluer la masse corporelle (GM). </li></ol> 4. Calculs <ol><li> Muscle: ratio de masse corporelle = masse musculaire / masse corporelle </li><li> Tensions Twitch, P t (Mn) = tension maximale produite lors de contraction </li><li> Tensions contraction spécifique (N / cm 2) = tensions contraction (mN) / section transversale (um 2) x 10 5 N / mN • um 2 / cm 2 </li><li> Le temps de la tension de crête (ms) = le temps entre l&#39;apparition de la contraction de la tension maximale </li><li> Demi-temps de relaxation (ms) = le temps de la tension crête à 50% de la tension maximale </li><li> La tension tétanique, P O (mn) = tension maximale générée pendant le tétanos </li><li> Spécifiques tensions tétaniques (N / cm 2) = tensions tétaniques (mN) / section transversale (um 2) x 10 5 N / mN • um 2 / cm 2 </li><li> Vitesse maximale de montée du tétanos (N / s) = taux maximum d&#39;augmentation de la tension pendant la montée des tensions dans le tétanos, c&#39;est à dire, la pente maximale de la courbe de la tension tétanique (ou, dP S / dt) </li><li> Tensions tétaniques demi-relaxation (ms) = le temps de la cessation de la stimulation à 50% de la tension à la cessation de la stimulation </li><li> Twitch tension au-tétanique ratio de tension, P t / P = O maximale de tension tétanique contraction de tension / maximum </li><li> Indice Fatigue = rapport de la tension après deux minutes de la fatigue de basse fréquence de la tension isométrique maximale </li></ol> 5. Les résultats représentatifs <img src="/files/ftp_upload/2582/2582fig1.jpg" alt="Figure 1" /> Figure 1. La dissection de muscle EDL. Une exposition, des membres postérieurs muscles.TA, jambier antérieur. B, exposition de l&#39;EDL (extensor digitorum longus) musculaire. C, la fixation des points de suture aux tendons EDL. D, bain capteur de tension (vue de côté). E, EDL montés dans le bain (vue de dessus). Le muscle de façon incomplète immergé dans la mémoire tampon à des fins d&#39;illustration, dans la pratique, le muscle doit être complètement immergé pour éviter de se dessécher. <img src="/files/ftp_upload/2582/2582fig2.jpg" alt="Figure 2" /> Figure 2. Exemple de courbes de tension. Un exemple, la courbe de la tension contraction illustrant la tension maximale contraction (P t), le temps de contraction (CT) et des demi-temps de relaxation (THS). Bar, 1s. B, Exemple de courbe de la tension tétanique maximale montrant la tension tétanique (P o) et tensions tétaniques demi-relaxation (HRTT). Bar, 1s. <img src="/files/ftp_upload/2582/2582fig3.jpg" alt="Figure 3" /> Figure 3. Exemple d&#39;analyse de la relation force-fréquence. Un tensions, fréquences générées par une stimulation supplémentaire. B, Exemple de train d&#39;impulsions à 30MHz. Bar, 80ms. C, Exemple de train d&#39;impulsions à 140MHz. Bar, 80ms. D, Exemple de force-fréquence courbe dérivée de données affichées dans un. La forme de la courbe force-fréquence est caractéristique de la force musculaire, et peut être comparé entre les muscles de différents animaux. <img src="/files/ftp_upload/2582/2582fig4.jpg" alt="Figure 4" /> Figure 4. Exemple d&#39;analyse de fréquence sans fatigue. Un tensions, décrémentielle généré plus de période de stimulation.Examples faible fréquence des trains d&#39;impulsions à des moments indiqué (B, C, D) sont indiqués ci-dessous. E Exemple de courbe de fréquence de fatigue faibles issus de données affichées dans un. La forme de la courbe de la fatigue de basse fréquence est caractéristique de la force musculaire, et peut être comparé entre les muscles de différents animaux.

<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>

Nous décrivons une approche détaillée afin d&#39;évaluer les propriétés mécaniques du myo-dans le muscle des membres postérieurs explantés de la souris. L&#39;EDL, tandis que plus difficiles à disséquer en raison de sa position postérieure derrière le muscle tibial antérieur, est plus facile à évaluer que le jambier antérieur en raison de ses éminents pièces jointes tendineux à la cheville et du genou. Ces tendons faciliter le montage dans la bande de myographe musculaire. En revanche, les plus facilement accessibles jambier antérieur a une large attachement presque atendinous à l&#39;articulation du genou, ce qui rend extrêmement difficile à la fois à décortiquer, sans compromettre le muscle, et monter en toute sécurité dans le myographe. Nous rappelons également que le montage rapide du muscle dans un bain oxygéné dans un tampon physiologique et la température est essentielle pour préserver les propriétés mécaniques du muscle. Nous avons constaté que nous pouvons répéter cette analyse pour un maximum de 30 minutes sans changements importants dans la réponse musculaire dans ces conditions. Enfin, il est essentiel que les fibres musculaires ne pas être touché lors de la dissection et les procédures de montage, comme cela peut avoir des effets néfastes sur la fonction musculaire, et aboutir à une sous-estimation de la myo-force mécanique. En suivant ces procédures, cette analyse offre une approche robuste quantitatives pour évaluer les effets de la modification génétique sur 6,7,8,9 fonction musculaire, ainsi que la comparaison entre les interventions thérapeutiques dans des modèles murins de la maladie du muscle 1,2,3,5 .

Solutions et Media Solution de Ringer lactate <ul><li> 100 mM de NaCl </li><li> 30 mM CH3CH (OH) COONa (lactate de sodium) </li><li> 4 mM de KCl </li><li> 1 mM CaCl 2, 2H 2 O (chlorure de calcium dihydraté) <ul><li> ajuster le pH à 6,75 </li></ul></li></ul> Krebs Henseleit solution <ul><li> 118 mM de NaCl </li><li> 4,7 mM de KCl </li><li> 1,25 mM de CaCl2 </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 glucose <ul><li> ajuster le pH à 7.2 à 7.4 en équilibrant avec O 2 / CO 2 (95% / 5%) de gaz </li></ul></li></ul> Pentobarbital <ul><li> 5 mg / ml, solution de travail dans l&#39;eau stérile </li></ul>

myo-mechanical analysis of isolated skeletal muscle

Pour évaluer les effets in vivo d&#39;interventions thérapeutiques pour le traitement de la maladie du muscle 1,2,3, méthodes quantitatives sont nécessaires pour mesurer et de génération de force dans les muscles traités fatigabilité. Nous décrivons une approche détaillée afin d&#39;évaluer les propriétés mécaniques du myo-dans le muscle des membres postérieurs fraîchement explantées de la souris. Nous décrivons la récolte atraumatique de la souris extenseurs muscle long fléchisseur, le montage du muscle dans une bande de muscle myographe (Modèle 820MS; danoises technologie Myo), et la mesure de la contraction maximale et la tension tétanique, le temps de contraction, et la demi-temps de relaxation, en utilisant un carrés d&#39;impulsion stimulateur (modèle S48; Technologies Grass). Grâce à ces mesures, nous démontrons le calcul de la contraction spécifiques et les tensions musculaires tétaniques normalisée à coupe transversale, la contraction-à-tétaniques ratio de la tension, la courbe de la relation force-fréquence et la courbe de la fatigue de basse fréquence 4. Cette analyse fournit une méthode de comparaison quantitative entre les interventions thérapeutiques dans des modèles murins de la maladie du muscle 1,2,3,5, ainsi que la comparaison des effets de la modification génétique sur la fonction musculaire 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

Pour évaluer la<em&gt; In vivo</em&gt; Les effets des interventions thérapeutiques pour les maladies musculaires, des méthodes sont nécessaires pour quantifier la force et la fatigabilité du muscle traité. Nous détaillons une approche pour évaluer les propriétés mécaniques du myo-dans le muscle de souris hindlimb explantés. Cette analyse fournit une approche robuste pour quantifier les effets de la modification génétique sur la fonction musculaire, ainsi que la comparaison des thérapies dans des modèles murins de maladies musculaires.

Myo-mécanique Analyse des muscles squelettiques isolés

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

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Medicine

26.7K vues.  University of California San Francisco. Pour évaluer la In vivo Les effets des interventions thérapeutiques pour les maladies musculaires, des méthodes sont nécessaires pour quantifier la force et la fatigabilité du muscle traité. Nous détaillons une approche pour évaluer les propriétés mécaniques du myo-dans le muscle de souris hindlimb explantés. Cette analyse fournit une approche robuste pour quantifier les effets de la modification génétique sur la fonction musculaire, ainsi que...

Vidéo: 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.

Skeletal Muscle dimorphisme des sexes de la protéomique

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

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Médecine

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;

Tibial Nerve Transection - un modèle standardisé pour dénervation induit l&#39;atrophie musculaire chez les souris

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.

Procédures Muscle squelettique humain biopsie aide de la technique de modification Bergström

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.

Préparation et respirométrique évaluation des mitochondries isolées de tissu musculaire squelettique Obtenu par biopsie percutanée aiguille

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.

Quantitative Imagerie par résonance magnétique de la maladie du muscle squelettique

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.

Humain<em&gt; Vaste externe</em&gt; Biopsie de muscle squelettique Utilisation de la 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.

Induction et évaluation des Exertional Skeletal dommages musculaires chez les humains

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.

Analyse semi-automatique de souris Muscle squelettique morphologie et la Composition de la fibre de type

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.

Couplage neurovasculaire Muscle squelettique, la capacité oxydative et microvasculaire fonction avec « One Stop Shop » de spectroscopie proche infrarouge

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.