Questo lavoro è stato sostenuto da un Pubblico Servizio Sanitario Grant (HL086513) dal NHLBI a PEO, e un assegno di ricerca completa del California Institute per la medicina rigenerativa (RC1-00104), un Pubblico Servizio Sanitario Grant (HL085377) da NHLBI, e un regalo dalla Fondazione Pollin di HSB SC è stato sostenuto da un Istituto della California per Ponti Medicina Rigenerativa di cellule staminali Research Award (TB1-01194) a San Francisco State University.

Il protocollo è eseguita con l&#39;approvazione del Institutional Animal Care UCSF e Comitato uso (IACUC). 1. Dissezione del mouse Longus estensore delle dita (EDL) muscolare <ol><li> Eseguire tutte le procedure animali in conformità con le linee guida istituzionali. </li><li> Eutanasia animale con 200 mg / kg intraperitoneale pentobarbital / cervicale dislocazione appena prima del raccolto del muscolo 10. La dissezione deve essere ben praticata in modo che il muscolo può essere raccolto e montato nel trasduttore di tensione entro 15 minuti di eutanasia. </li><li> Disporre in posizione supina carcassa sul vassoio dissezione e la gamba pin vassoio. </li><li> Sotto tibiale microscopio dissezione, la pelle aperto, fascia aprire con cautela (Fig.1a), e la buccia dalla caviglia verso l&#39;alto per esporre EDL (Fig.1B). Utilizzare gocce di soluzione di Ringer lattato per mantenere il muscolo umido e tamponata durante la vendemmia. </li><li> Rimuovere EDL, conservando il tendine il più possibile su ogni estremità, e messo in una capsula di Petri contenente soluzione di Ringer lattato. Tie sutura a ciascuno dei tendini dei muscoli (Fig.1C). E &#39;essenziale che le fibre muscolari non essere toccato o disturbato durante la dissezione. </li></ol> 2. Montaggio del EDL Mouse nel miografo Striscia muscolare <ol><li> Per questi studi, un bagno di tessuto è necessario che mantiene i muscoli consentendone al contempo fare il bagno in soluzione fisiologica a temperatura costante con l&#39;ossigenazione continuo. Il bagno è accoppiato con un trasduttore di forza per la misura della tensione muscolare. Ci avvaliamo di un sistema integrato di fascia muscolare bagno miografo dal danese Myo Technology (DMT modello 820MS) per questo scopo. Inoltre, uno stimolatore elettrico impulso quadrato (Grass modello S48) e la piattaforma di acquisizione dati (ADInstruments PowerLab sistema di acquisizione dati e software LabChart) sono necessari per sollecitare, registrare e analizzare mio-meccanico risposte, rispettivamente. Il 820MS DMT ha elettrodi di platino integrato nel coperchio della camera che sono posizionati su entrambi i lati del muscolo, a metà porzione della striscia muscolare. Myographs altri possono richiedere una specifica attenzione al posizionamento degli elettrodi. </li><li> Riempi bagno miografo con 5 mL di soluzione Krebs Henseleit 11. Calda a 25 ° C. Bubble O 2 / CO 2 (95% / 5%) attraverso il bagno per 15 minuti prima dell&#39;uso. </li><li> Utilizzare punti di sutura per estendere EDL tra i morsetti di miografo e sicuro i tendini del muscolo EDL (Fig.1D, E) tra i morsetti. Fare attenzione a non bloccare il muscolo stesso. </li><li> Mantenere miografo bagno a 25 ° C. </li></ol> 3. Myo-meccanico Analisi A. Twitch tensione <ol><li> Imposta lunghezza iniziale nel bagno in modo che non ci sia lassismo muscolare. </li><li> Determinare stimolo massima (durata 0,5 ms) regolando tensione al fine di ottenere la tensione massima contrazione, quindi impostare stimolo al 20% al di sopra massima (per raggiungere stimolo sovramassimale). Nei nostri studi, lo stimolo sovramassimale di solito è raggiunta a una potenza di 40 volt. </li><li> Verificare uscita dallo stimolatore con un oscilloscopio. </li><li> Stabilire la lunghezza ottimale gradualmente il muscolo che si estende fino a quando non vi è ulteriore aumento della tensione contrazione. </li><li> Lasciare muscolo equilibrare per 3 min. </li><li> Fornire sovramassimale stimolo quadrati (0,5 ms) a lunghezza ottimale utilizzando Erba S44 stimolatore elettronico, e l&#39;uscita record. </li><li> Record: la tensione della curva contrazione (P vs tempo t; Fig.2a). </li></ol> Tetano B. tensione <ol><li> Lasciare muscolare a riposo per 3 minuti. </li><li> Applicare un treno di stimoli sovramassimale per 300msec a 150 Hz a lunghezza ottimale utilizzando Erba S44 stimolatore elettronico, e l&#39;uscita record. </li><li> Record: la tensione della curva tetano (P o vs tempo; Fig.2b). </li></ol> C. Forza frequenza <ol><li> Lasciare muscolare a riposo per 3 minuti. </li><li> Forza-frequenza: applicare i treni di stimoli sovramassimale a 30, 60, 100, 140 e 160 Hz con 3 minuti di riposo tra ogni stimolo (Fig.3). In alternativa, i treni possono essere applicati 15, 25, 35, 45, 55, 65, 75, 100, 140 e 160 Hz per una migliore risoluzione a frequenze più basse, dove i cambiamenti forza in modo sostanziale. </li><li> Trama: forza-frequenza di rapporti (% vs frequenza massima forza di stimolazione). </li></ol> D. fatica <ol><li> Applicare treno di brevi tetani: 60 Hz per 300 ms (o la frequenza regolare di produrre il 50% della forza di picco), ogni 3 secondi per 10 minuti. Da 10 minuti, la forza tetanica dovrebbe ridursi ad un livello di plateau di circa il 15% del valore iniziale (Fig. 4). </li><li> Trama: fatica a bassa frequenza (% massima rispetto al tempo vigenti). </li></ol> E. la raccolta di dati aggiuntivi alla fine del protocollo <ol><li> Prima di smontare il muscolo dal miografo, impostare il muscle in lunghezza ottimale, come determinato al punto III.A.4 e misurare il suo diametro utilizzando un oculare del microscopio o con le pinze. Calcolare sezione trasversale (micron 2). </li><li> Misurare la massa muscolare (mg) togliendo punti di sutura e un peso muscolare. </li><li> Pesare mouse per valutare la massa corporea (gm). </li></ol> 4. Calcoli <ol><li> Muscolare: massa rapporto corpo = massa muscolare / massa corporea </li><li> Twitch tensione, P t (mN) = massima tensione generati durante la contrazione </li><li> Contrazione tensione specifica (N / cm 2) = contrazione tensione (mN) / sezione trasversale (micron 2) x 10 5 N / mN • micron 2 / cm 2 </li><li> Tempo per la tensione di picco (ms) = tempo dalla comparsa della contrazione di massima tensione </li><li> A metà tempo di rilassamento (ms) = tempo dalla tensione di picco al 50% della tensione di picco </li><li> Tetanica tensione, P O (mn) = massima tensione generati durante il tetano </li><li> Specifica tensione tetanica (N / cm 2) = tetanica tensione (mN) / sezione trasversale (micron 2) x 10 5 N / mN • micron 2 / cm 2 </li><li> Tasso massimo di aumento del tetano (N / s) = tasso massimo di aumento della tensione durante l&#39;ascesa tensione nel tetano, vale a dire la massima pendenza della curva di tensione tetanica (o, O dP / dt) </li><li> Mezza rilassamento della tensione tetanica (ms) = tempo dalla cessazione della stimolazione al 50% della tensione al termine della stimolazione </li><li> Twitch tensione-to-tetanica rapporto di tensione, P t / P O = contrazione massima tensione / massima tensione tetanica </li><li> Indice di stanchezza = rapporto di tensione dopo due minuti di fatica a bassa frequenza per raggiungere la massima tensione isometrica </li></ol> 5. Rappresentante Risultati <img src="/files/ftp_upload/2582/2582fig1.jpg" alt="Figura 1" /> Figura 1. Dissezione del muscolo EDL. A, esposizione di arti posteriori muscles.TA, tibiale anteriore. B, esposizione di EDL (dell&#39;estensore lungo delle dita) del muscolo. C, allegato di suture di tendini EDL. D, trasduttore bagno Tensione (vista dal lato). E, EDL montato in bagno (vista dall&#39;alto). Il muscolo in modo incompleto immerso nel buffer a scopo illustrativo, in pratica, il muscolo deve essere completamente immerso per evitare che si secchi. <img src="/files/ftp_upload/2582/2582fig2.jpg" alt="Figura 2" /> Figura 2. Esempio di curve di tensione. A, Esempio di curva di contrazione tensione illustrando tensione massima contrazione (P t), tempo di contrazione (CT) e metà tempo di rilassamento (HRT). Bar, 1s. B, Esempio di curva tensione tetanica mostrando massima tensione tetanica (P o) e mezzo di rilassamento della tensione tetanica (HRTT). Bar, 1s. <img src="/files/ftp_upload/2582/2582fig3.jpg" alt="Figura 3" /> Figura 3. Esempio di forza-frequenza analisi della relazione. A, le tensioni generate da frequenze di stimolazione incrementale. B, Esempio di treno di impulsi a 30MHz. Bar, 80ms. C, Esempio di treno di impulsi a 140 MHz. Bar, 80ms. D, Esempio di forza-frequenza curva derivata da dati riportati in A. La forma della curva forza-frequenza è caratteristica della forza muscolare, e possono essere confrontati tra i muscoli di animali diversi. <img src="/files/ftp_upload/2582/2582fig4.jpg" alt="Figura 4" /> Figura 4. Esempio di analisi a bassa frequenza fatica. A, le tensioni decrementale generato oltre stimulation.Examples periodo di bassa frequenza dei treni di impulsi a intervalli di tempo indicato (B, C, D) sono riportati di seguito. E, Esempio di curva a bassa frequenza stanchezza derivata da dati riportati in A. La forma della curva di fatica a bassa frequenza è caratteristica della forza muscolare, e possono essere confrontati tra i muscoli di animali diversi.

<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>

Nessun conflitto di interessi dichiarati.

Descriviamo un approccio dettagliato per valutare myo-meccaniche in espiantati muscolare degli arti posteriori dal mouse. L&#39;EDL, mentre più difficile da analizzare a causa della sua posizione posteriore dietro il muscolo tibiale anteriore, è più facile da valutare rispetto al tibiale anteriore a causa della sua allegati importanti tendinea alla caviglia e le articolazioni del ginocchio. Questi tendini facilitare il montaggio nel miografo fascia muscolare. Al contrario, tanto più facilmente accessibili tibiale anteriore ha un ampio, l&#39;attaccamento quasi atendinous al ginocchio, rendendo estremamente difficile sezionare entrambi senza compromettere il muscolo, e montare in modo sicuro nel miografo. Abbiamo, inoltre, che in rapida crescita il muscolo in un bagno ossigenato in un tampone fisiologico e la temperatura è essenziale per preservare le proprietà meccaniche del muscolo. Abbiamo scoperto che possiamo ripetere questa analisi per un massimo di 30 minuti senza significativi cambiamenti nella risposta muscolare in queste condizioni. Infine, è essenziale che le fibre muscolari non essere toccato durante la dissezione e le procedure di montaggio, in quanto ciò può avere effetti negativi sulla funzione muscolare e portare a una sottostima del mio-meccanico forza. Seguendo queste procedure, questa analisi fornisce un approccio robusto quantitativo di valutare gli effetti delle modificazioni genetiche sulla funzione muscolare 6,7,8,9, così come il confronto tra interventi terapeutici in modelli murini di malattia muscolare 1,2,3,5 .

Soluzioni e Media Soluzione di Ringer lattato <ul><li> 100 mM NaCl </li><li> 30 mM CH3CH (OH) COONa (lattato di sodio) </li><li> 4 mM KCl </li><li> 1 mM CaCl 2 2H 2 O (cloruro di calcio diidrato) <ul><li> aggiustare il pH a 6,75 </li></ul></li></ul> Krebs Henseleit soluzione <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 di glucosio <ul><li> aggiustare il pH a 7,2-7,4 da equilibrare con O 2 / CO 2 (95% / 5%) del gas </li></ul></li></ul> Pentobarbital <ul><li> 5 mg / mL di soluzione di lavoro in acqua sterile </li></ul>

myo-mechanical analysis of isolated skeletal muscle

Per valutare gli effetti in vivo degli interventi terapeutici per il trattamento della malattia muscolare 1,2,3, sono necessari metodi quantitativi che misurano costituzione della forza e affaticamento nei muscoli trattati. Descriviamo un approccio dettagliato per valutare myo-meccaniche in appena espiantato muscolare degli arti posteriori dal mouse. Descriviamo la raccolta atraumatica di longus topo muscolo estensore comune delle dita, il montaggio del muscolo in un muscolo striscia miografo (Modello 820MS; danese Tecnologia Myo), e la misurazione della contrazione massima e tensione tetanica, tempo di contrazione e rilassamento a metà tempo, utilizzando un piazza impulso stimolatore (modello S48; Technologies Grass). Utilizzando queste misure, dimostriamo il calcolo di specifici contrazione e la tensione muscolare tetanica normalizzata a sezione trasversale, il tic-to-tetanica rapporto di tensione, la forza-frequenza curva di relazione e la bassa curva di fatica frequenza 4. Questa analisi fornisce un metodo per il confronto quantitativo tra interventi terapeutici in modelli murini di malattia muscolare 1,2,3,5, così come il confronto degli effetti delle modificazioni genetiche sulla funzione muscolare 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

Per valutare l&#39;<em&gt; In vivo</em&gt; Effetti degli interventi terapeutici per le malattie muscolari, i metodi sono necessari per quantificare costituzione della forza e affaticamento nei muscoli trattati. Noi dettaglio un approccio alla valutazione myo-meccaniche nel muscolo del mouse espiantati arti posteriori. Questa analisi fornisce un valido approccio per la quantificazione degli effetti delle modificazioni genetiche sulla funzione muscolare, così come il confronto di terapie in modelli murini di malattia muscolare.

Myo-meccanico Analisi dei isolata del muscolo scheletrico

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 Views.  University of California San Francisco. Per valutare l' In vivo Effetti degli interventi terapeutici per le malattie muscolari, i metodi sono necessari per quantificare costituzione della forza e affaticamento nei muscoli trattati. Noi dettaglio un approccio alla valutazione myo-meccaniche nel muscolo del mouse espiantati arti posteriori. Questa analisi fornisce un valido approccio per la quantificazione degli effetti delle modificazioni genetiche sulla funzione muscolare, così come il confr...

Video: 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.

Sesso Dimorfismo muscolo scheletrico da Proteomica

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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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;

Resezione del nervo tibiale - Un modello standardizzato per Denervation indotta scheletrico atrofia muscolare nei topi

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.

Scheletrici Procedure biopsia muscolare dell&#39;uomo con la tecnica Bergström Modificato

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.

Preparazione e respirometrici valutazione dei mitocondri isolati dal tessuto muscolare scheletrico Ottenuto dalla biopsia percutanea

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 Magnetic Resonance Imaging di malattia muscolare scheletrico

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.

Umano<em&gt; vasto laterale</em&gt; Biopsia muscolare scheletrico Utilizzando 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.

Induzione e valutazione di Exertional scheletrico danno muscolare negli esseri umani

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.

Analisi semi-automatica del Mouse del muscolo scheletrico morfologia e composizione di 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.

Muscolo scheletrico Neurovascular accoppiamento, capacità ossidativa e funzione microvascolare con 'One Stop Shop' spettroscopia nel vicino infrarosso

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.