Bu çalışma NHLBI PEO için Halk Sağlığı Servisi Grant (HL086513) ve Rejeneratif Tıp (RC1-00104), Kamu Sağlık Hizmetleri NHLBI Grant (HL085377) için California Institute Kapsamlı Araştırma Fonu tarafından desteklenen ve bir hediye Pollin&#39;in Vakfı HSB SC Kök Hücre Araştırma Ödülü San Francisco State University (TB1-01.194) bir California Rejeneratif Tıp Köprüler Enstitüsü tarafından desteklenmiştir.

Protokol UCSF Kurumsal Hayvan Bakım ve Kullanım Kurulu (IACUC) onayı ile yapılır. 1. Fare ekstansör digitorum longus (EDL) kas diseksiyonu <ol><li> Kurumsal kurallarına uygun olarak tüm hayvan işlemleri gerçekleştirin. </li><li> 10 kas hasattan hemen önce 200 mg / kg intraperitoneal / pentobarbital servikal dislokasyon ile hayvan Euthanize. Diseksiyonu iyi ötenazi 15 dakika içinde kas hasat ve gerilim dönüştürücü monte edilebilir şekilde uygulanmalıdır. </li><li> Diseksiyonu tepsisi ve tepsiye pin bacak karkas yatar düzenleyin. </li><li> EDL (Fig.1B) ayak bileği diseksiyon mikroskobu, açık cilt, dikkatli bir şekilde açık fasya (Fig.1A), ve kabuğu tibialis altında açığa çıkarmak için yukarı. Kas nemli ve hasat sırasında tamponlu tutmak Laktatlı Ringer solüsyonu damla kullanın. </li><li> Her iki ucunda, mümkün olduğu kadar çok tendon olarak koruyarak, EDL çıkarın ve Laktatlı Ringer çözeltisi içeren Petri koymak. Kravat dikiş her kas tendon (Fig.1C). Bu kas lifleri dokunulduğunda veya diseksiyon sırasında rahatsız edilmemesi esastır. </li></ol> 2. Kas Şeridi&#39;nde Myograph Fare EDL montajı <ol><li> Bu çalışmalar için, bir doku banyosu sürekli oksijenasyonu ile sabit sıcaklıkta fizyolojik çözüm yıkanmasına izin verirken kas güvence altına ihtiyaç vardır. Banyo, kas gerginliği ölçümü için bir güç dönüştürücü ile birleştirilmiştir. Biz bu amaç için Danimarka Myo Teknolojisi (DMT Model 820MS) entegre bir kas şeridi myograph banyo kullanırlar. Buna ek olarak, bir kare dalga, elektrik stimülatörü (Grass Model S48) ve veri toplama platformu (ADInstruments PowerLab Veri Toplama Sistemi ve LabChart yazılım), sırasıyla, myo-mekanik yanıtları kaydetmek ve analiz ortaya çıkarmak için ihtiyaç vardır. DMT 820MS kas şeridinin orta kısmı, kas iki tarafında konumlandırılmış bölmesi kapağı, entegre platin elektrotlar vardır. Diğer myographs elektrot yerleştirme için özel dikkat gerektirebilir. </li><li> 5 ml Krebs Henseleit solüsyonu 11 ile myograph banyo doldurun. 25 ° C sıcak Kullanmadan önce 15 dakika boyunca banyo ile Bubble O 2 / CO 2 (% 95 /% 5). </li><li> Myograph, kelepçeler arasında EDL genişletmek ve kelepçeler arasında EDL kas ve tendonların (Fig.1D E) güvence altına almak için dikişlerle kullanın. Kas kendisine kelepçe için dikkatli olun. </li><li> 25 myograph banyo koruyun ° C. </li></ol> 3. Myo-mekanik Analizi A. Twitch gerginlik <ol><li> Banyo ilk uzunluğu, kas gevşekliği olacak şekilde ayarlayın. </li><li> Sonra maksimal seğirme gerilim elde etmek için gerilimi ayarlayarak maksimum uyaran (süresi 0,5 ms) belirleyin,% 20 maksimum (supramaksimal uyaran ulaşmak için) uyaran ayarlanır. Çalışmalarda, supramaksimal uyaran genellikle 40 volt bir çıkış elde edilir. </li><li> Stimülatörü bir osiloskop kullanarak çıkış olun. </li><li> Seğirme gerginliği daha fazla bir artış kalmayıncaya kadar kademeli olarak kas germe optimal uzunluğunu belirlemek. </li><li> 3 dakika kas denge izin ver. </li><li> Çim S44 elektronik stimülatör kullanarak optimum uzunlukta supramaksimal kare uyaran (0.5 ms) ve kayıt çıkış sunun. </li><li> Kayıt: seğirme gerilim eğrisi (P t-zaman; Fig.2A). </li></ol> B. Tetanoz gerginlik <ol><li> Kas 3 dakika dinlenmek için izin ver. </li><li> Çim S44 elektronik stimülatör kullanarak optimum uzunlukta 150 Hz 300msec supramaksimal uyaranlara tren, ve kayıt çıkış uygulayın. </li><li> Rekor: tetanoz gerilim eğrisi (P o-zaman; Fig.2B). </li></ol> C. Force frekans <ol><li> Kas 3 dakika dinlenmek için izin ver. </li><li> Kuvvet-frekansı: her uyarıcı (Şekil 3) arasında kalan 3 dakika ile 30, 60, 100, 140, ve 160 Hz supramaksimal uyaranların trenlerde geçerli. Alternatif olarak, tren, 15, 25, 35, 45, 55, 65, 75, 100, 140 ve daha düşük frekanslarda daha iyi çözünürlük için 160 Hz kuvveti değişiklikler büyük ölçüde uygulanabilir. </li><li> Konu: kuvvet-frekans ilişkisi (% maksimal kuvvet vs stimülasyon frekansı). </li></ol> D. Yorgunluk <ol><li> 10 dakika için 300 ms (veya% 50 maksimum kuvvet üretmek için ayarlanabilir frekans), her 3 saniyede 60 Hz kısa tetani tren uygulayın. 10 dakika, tetanik kuvvetin başlangıç ​​değeri (Şekil 4) ~% 15 oranında bir plato seviyesine inmesi gerekir. </li><li> Konu: düşük frekans yorgunluk (% maksimal kuvvet-zaman). </li></ol> E. protokol sonunda Ek veri toplama <ol><li> Myograph kas nasıl açıp kapayabilirsiniz önce musc ayarlamakle adım III.A.4 belirlenen optimal uzunlukta ve mikroskop veya kaliperler ile ya bir göz kullanarak kendi çapını ölçün. Kesit alanı (mikron 2) hesaplayın. </li><li> Sütür kaldırılması ve tartım kas kas kütlesi (mg) ölçün. </li><li> Vücut kütlesi (gr) değerlendirmek için fare tartılır. </li></ol> 4. Hesaplamalar <ol><li> Muscle: vücut kütle oranı = kas kütlesi / vücut kitle </li><li> Twitch gerginlik, P t (MN) = seğirme sırasında oluşan maksimum gerilimi </li><li> Özgül seğirme gerginliği (N / cm 2) = seğirme gerilimi (mN) / kesit alanı (mikron 2) x 10 5 N / mN • mikron 2 / cm 2 </li><li> Pik gerilimi Süresi (ms) = maksimum gerilim daralma başlaması zaman </li><li> Half-gevşeme zamanı (ms) = pik gerilimi tepe gerilim% 50 zaman </li><li> Tetanik gerginlik, P O (MN) = tetanoz sırasında oluşan maksimum gerilimi </li><li> Özel tetanik gerginliği (N / cm 2) = tetanik gerilimi (mN) / kesit alanı (mikron 2) x 10 5 N / mN • mikron 2 / cm 2 </li><li> Tetanoz yükselişi maksimal hızı (KB / s) = tetanoz, yani tetanik gerilim eğrisi (veya dP O / dt) maksimum eğim gerginlik yükselişi sırasında gerginlik artışı maksimum hızı </li><li> Half-rahatlama tetanik gerilimi (ms) = stimülasyon bırakma stimülasyon bitiminde gerginlik% 50 zaman </li><li> Twitch gerilim-tetanik gerilim oranı, P t / P O = maksimum seğirme gerilim / maksimum tetanik gerginlik </li><li> Yorgunluk indeksi = maksimal izometrik gerilim düşük frekanslı yorgunluk iki dakika sonra gerginlik oranı </li></ol> 5. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2582/2582fig1.jpg" alt="Şekil 1" /> Şekil 1. EDL kas diseksiyonu. Hindlimb muscles.TA, tibialis anterior B, EDL (ekstansör digitorum longus) kas Pozlama, Pozlama C, EDL tendonları sütür Ek D, Gerilim transdüser banyo (yandan görünüm) E, EDL banyo monte (üstten görünüm). Kas eksik amaçlıdır tampon dalmış, pratikte, kas tamamen kurumasını önlemek için dalmış olmalıdır. <img src="/files/ftp_upload/2582/2582fig2.jpg" alt="Şekil 2" /> Şekil 2. Örnek gerilim eğrileri. Seğirme gerilim eğrisinin maksimum seğirme gerilimi (P t), kasılma süresi (BT) ve yarı-gevşeme zamanı (HRT) gösteren A, Örnek Bar, 1s B, maksimal tetanik gerginlik gösteren tetanik gerilim eğrisi (P o) Örnek ve yarım rahatlama tetanik gerilimi (HRTT). Bar, 1s. <img src="/files/ftp_upload/2582/2582fig3.jpg" alt="Şekil 3" /> Şekil 3. Örnek, kuvvet-frekans ilişkisi analizi . Artımlı stimülasyon frekansları tarafından oluşturulan bir Gerginlikler B, 30MHz puls Örnek Bar, 80ms C, 140MHz puls örneği. Bar, 80ms D, A gösterilen veri elde edilen kuvvet-frekans eğrisi örneği. Kuvvet-frekans eğrisi şekli kas gücü karakteristik ve farklı hayvanların kasları arasında mukayese edilebilir. <img src="/files/ftp_upload/2582/2582fig4.jpg" alt="Şekil 4" /> Şekil 4. Düşük frekanslı yorgunluk analizi örneği. , Belirtilen zaman noktalarında (B, C, D) nabız trenler düşük frekanslı stimulation.Examples süre içinde oluşturulan, azalan gerilimleri aşağıda gösterildiği E, A gösterilen veri elde edilen düşük frekanslı yorgunluk eğrisi örneği . Düşük frekanslı yorgunluk eğrisinin şekli kas gücü karakteristik ve farklı hayvanların kasları arasında mukayese edilebilir.

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	<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>

Biz fare eksplante hindlimb kas myo-mekanik özelliklerinin değerlendirilmesi için ayrıntılı bir yaklaşım açıklar. EDL, tibialis anterior kas arkasında posterior pozisyon nedeniyle incelemek için daha zor iken, tibialis anterior önemli tendinöz ekleri nedeniyle ayak bileği ve diz eklemleri daha değerlendirmek için daha kolaydır. Bu tendonlar, kas şeridi myograph montajı kolaylaştırmak. Aksine, daha kolay erişilen tibialis anterior derece zor hem kas ödün vermeden teşrih ve myograph güvenli bir şekilde monte, geniş bir diz eklemi, neredeyse atendinous eki vardır. Ayrıca fizyolojik tampon ve sıcaklık hızla oksijenli hamamı kas montaj kas mekanik özelliklerinin korunması için gerekli olduğuna dikkat çekiyor. Biz, biz, bu koşullar altında kas yanıt olarak önemli değişiklikler olmadan 30 dakika kadar bu analiz tekrarlayabilirsiniz bulduk. Sonuç olarak, kas lifleri, kas fonksiyonu üzerinde olumsuz etkileri var ve myo-mekanik güç, küçümsenmesi sonucu olarak diseksiyon ve montaj prosedürleri sırasında dokunulmaz değil esastır. Bu prosedürler izlenerek, bu analiz 6,7,8,9 kas fonksiyonu genetik modifikasyon etkileri yanı sıra, kas hastalıkları 1,2,3,5 fare modelleri terapötik müdahaleler arasında karşılaştırma değerlendirilmesi için sağlam bir nicel bir yaklaşım sağlar .

Çözümler ve Medya Ringer solüsyonu <ul><li> 100 mM NaCl </li><li> 30 mM CH3CH (OH) COONa (sodyum laktat) </li><li> 4 mM KCl </li><li> 1 mM CaCl 2 2H 2 O (kalsiyum klorür dihidrat) <ul><li> 6.75 ile pH ayarı </li></ul></li></ul> Krebs Henseleit çözümü <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 glukoz <ul><li> O 2 / CO 2 (% 95 /% 5) gaz ile değişikti tarafından 7,2-7,4 pH ayarı </li></ul></li></ul> Pentobarbital <ul><li> Steril su içinde 5 mg / ml çalışma çözüm </li></ul>

myo-mechanical analysis of isolated skeletal muscle

1,2,3 kas hastalığının tedavisi için terapötik girişimlerin in vivo etkileri değerlendirmek için, kantitatif yöntemlere ihtiyaç tedavi kas kuvvet üretimi ve yorulabilirlik önlem. Biz fare taze eksplante hindlimb kas myo-mekanik özelliklerinin değerlendirilmesi için ayrıntılı bir yaklaşım açıklar. Kullanarak, maksimum seğirme ve tetanik gerginlik, kasılma süresi, ve yarım gevşeme zamanı ölçme; kas kas şeridi myograph (Danimarka Myo Teknoloji Model 820MS) montaj, fare ekstansör digitorum longus kas atravmatik hasat tarif kare dalga Stimülatörü (Model S48, Çim Teknolojileri). Bu ölçümler kullanılarak, kas kesit alanı, seğirme-tetanik gerilim oranı, kuvvet-frekans ilişkisi eğrisi ve düşük frekanslı yorgunluk eğrisi 4 normalize özel seğirme ve tetanik gerilim hesaplama göstermektedir. Bu analiz, tedavi fare modelleri, kas hastalıkları 1,2,3,5 müdahalelerin yanı sıra, kas fonksiyonu 6,7,8,9 genetik modifikasyon etkilerinin karşılaştırılması arasındaki sayısal karşılaştırma için bir yöntem sağlar .

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

Değerlendirmek için<em&gt; In vivo</em&gt; Etkileri, kas hastalıkları için tedavi edici girişimlerin yöntemleri tedavi edilen kas kuvvet üretimi ve yorulabilirlik ölçmek için ihtiyaç vardır. Biz ayrıntılı eksplante fare hindlimb kas myo-mekanik özelliklerini değerlendiren bir yaklaşım. Bu analiz, kas fonksiyonu genetik değişiklik, kas hastalığı fare modelleri tedavilerin yanı sıra karşılaştırma etkileri quantitating için sağlam bir yaklaşım sağlar.

İzole İskelet Kası Myo-mekanik Analizi

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

Nanyang Technological University

Research

JoVE Journal

Medicine

26.7K Görüntüleme.  University of California San Francisco. Değerlendirmek için In vivo Etkileri, kas hastalıkları için tedavi edici girişimlerin yöntemleri tedavi edilen kas kuvvet üretimi ve yorulabilirlik ölçmek için ihtiyaç vardır. Biz ayrıntılı eksplante fare hindlimb kas myo-mekanik özelliklerini değerlendiren bir yaklaşım. Bu analiz, kas fonksiyonu genetik değişiklik, kas hastalığı fare modelleri tedavilerin yanı sıra karşılaştırma etkileri quantitating için sağlam bir yaklaş...

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.

İskelet Kası Cinsiyet Proteomik Dimorfizm

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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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 Sinir transeksiyonu - Farelerde denervasyon bağlı İskelet Kas Atrofi için bir Standart Model

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.

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

Modifiye Bergström Tekniği İnsan İskelet Kası Biyopsi Prosedürleri

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.

Perkütan İğne Biyopsisi ile Elde Edilen İskelet Kası Doku İzole Mitokondri hazırlanması ve Respirometrik Değerlendirmesi

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.

İskelet Kası Hastalığının kantitatif Manyetik Rezonans Görüntüleme

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

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Farelerde Basınç Aşırı Modeli Koroner Arter Akış ve Koroner Akım Rezervi Ultrason Dayalı Değerlendirme

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histolojik Analiz kas cryosections Hazırlanmasında Yapılması ve Yapılmaması Gerekenler

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology 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.

İnsan<em&gt; Vastus lateralis</em&gt; Weil-Blakesley Conchotome kullanılması İskelet Kası Biyopsi

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.

İndüksiyon ve İnsanlarda Egzersizde İskelet Kası Hasar değerlendirilmesi

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

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Fosfor-31 Manyetik Rezonans Spektroskopisi: Ölçme için Aracı<em&gt; İn Vivo</emİnsan İskelet Kası içinde&gt; Mitokondriyal Oksidatif Fosforilasyon Kapasitesi

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...