この作品は、NHLBIからPEOへの公衆衛生サービスグラント（HL086513）、および再生医療（RC1 - 00104）のためのカリフォルニア工科大学、NHLBIから公衆衛生サービスグラント（HL085377）、およびギフトから包括的な研究助成金によってサポートされていましたポリン財団からHSBへ SCは、サンフランシスコ州立大学への細胞の研究賞（TB1 - 01194）を食い止めるために再生医療の橋のためにカリフォルニア工科大学によってサポートされていました。

プロトコルは、UCSF動物実験の承認を得て実施し、委員会（IACUC）を使用しています。 1。マウス指伸筋の筋 （EDL）筋肉の解剖<ol><li>制度的なガイドラインに従って、すべての動物の手順を実行します。 </li><li>筋肉の収穫は10〜直前に200 mg / kgを腹腔内子宮頸部/ペントバルビタール脱臼で動物を安楽死させる。解剖はよく筋肉が安楽死の15分以内に収穫し、緊張の変換器でマウントできるように練習してください。 </li><li>解剖のトレイとトレイのピン脚の上で死体の仰臥位を手配する。 </li><li>解剖顕微鏡、開いている皮膚、慎重に開いている筋膜（ 図1A）、そして足首から脛骨皮の下にEDL（ 図1b）を公開する上方。筋肉がしっとりと収穫の間にバッファを維持するために乳酸リンゲル液の滴を使用してください。 </li><li> EDLを削除する、それぞれの端に、できるだけ多くの腱として維持し、及び乳酸リンゲル液を含むペトリ皿に入れ。筋肉の腱（Fig.1C）の各々に縫合糸を接続します。それは筋肉の繊維が触れたり、解剖時に邪魔されないことが不可欠である。 </li></ol> 2。筋肉のストリップミオグラフでマウスのEDLの取付け<ol><li>これらの研究のために、組織の風呂は、それが連続的な酸素化と一定の温度で生理的溶液中で入浴しながらことは筋肉を固定して必要とされる。お風呂は、筋肉の緊張の測定のための力変換器で結合されている。我々はこの目的のためにデンマークのミオテクノロジー（DMTモデル820MS）から統合された筋肉のストリップミオグラフバスを採用しています。さらに、方形パルス電気刺激装置（グラスモデルS48）とデータ収集プラットフォーム（ADInstruments PowerLabデータ収録システムとLabChartソフトウェア）はそれぞれ、ミオ機械的応答を記録して分析、引き出すために必要とされる。 DMT 820MSは、筋肉のストリップの中間部分で、筋肉の両側に配置されているチャンバーカバーに統合された白金電極を持っています。他のmyographsは、電極の配置に固有の注意が必要な場合があります。 </li><li> 5mLのクレブスヘンゼライト溶液11にミオグラフバスを埋める。 25℃くらいまで温め使用前に15分間入浴を通じてバブルO 2 / CO 2（95％/ 5％）。 </li><li>ミオグラフのクランプ間にEDLを拡張し、クランプ間EDL筋肉の腱（Fig.1D、E）を確保するために縫合糸を使用してください。筋肉自体を固定しないように注意してください。 </li><li> 25℃ミオグラフバスを維持℃に</li></ol> 3。ミオ - 機械的分析 A.の単収縮張力<ol><li>は筋肉の弛緩がないように槽内の初期の長さを設定します。 </li><li> 、最大の単収縮張力を得るために電圧を調整することによって、最大刺激（持続時間は0.5 ms）を決定するには、その後、最大上記の20％（最大上刺激を達成するために）で刺激を設定します。我々の研究では、最大上刺激は通常40ボルトの出力で達成される。 </li><li>オシロスコープを使って刺激からの出力を確認します。 </li><li>単収縮張力にはさらなる増加がなくなるまで徐々に筋肉をストレッチすることにより最適な長さを決定します。 </li><li>筋肉は3分間平衡させる。 </li><li>最大上の正方形グラスS44電気刺激装置を使用して最適な長さの刺激（0.5ミリ）、およびレコードの出力を提供します。 </li><li>レコード：単収縮張力曲線（P T対時間、Fig.2A）。 </li></ol> B.破傷風緊張<ol><li>筋肉は3分間休息することができます。 </li><li>グラスS44電気刺激装置を使用して最適な長さで150 Hzで300ミリ秒のための最大上刺激の列車、およびレコードの出力を適用する。 </li><li>レコード：破傷風の張力曲線（P O対時間、 図2b）。 </li></ol> C.フォース周波<ol><li>筋肉は3分間休息することができます。 </li><li>力周波数：それぞれの刺激（ 図3）の間に、残り3分で30、60、100、140、および160 Hzで最大上刺激の列車を適用する。また、電車は15、25、35、45、55、65、75、100、140と低い周波数での分解能は160 Hzの、力の変化が、実質的に適用されることがあります。 </li><li>プロット：力 - 周波数の関係（％最大力対刺激の周波数）。 </li></ol> D.疲労<ol><li>短い破傷風菌の列車を適用する：300ミリ秒（または周波数ピーク力の50％を生産するように調整）は60 Hz、10分間毎に3秒。 10分では、破傷風の力は初期値（ 図4）の〜15％のプラトーレベルに低下するはず。 </li><li>プロット：低周波疲労（％最大の力対時間）。 </li></ol>プロトコルの最後にE.追加のデータ収集<ol><li>ミオグラフから筋肉をアンマウントする前に、MUSCを設定するルは、最適な長さでステップIII.A.4で決定され、顕微鏡やキャリパーのいずれかで眼を使用して、その直径を測定するように。断面積（μmの2）を計算する。 </li><li>縫合糸を除去し、筋肉を比較検討することによって筋肉の質量（mg）を測定。 </li><li>本体質量（GM）を評価するためにマウスを量る。 </li></ol> 4。計算<ol><li>筋肉：ボディの質量比= 筋肉量/ボディ質量</li><li>単収縮張力、P T（MN）= けいれん時に生成される最大張力</li><li>特定の単収縮張力（N / cm 2）と= 単収縮張力（MN）/断面積（μm2と ）× 10 5 N / MN•μmの2 / cm 2で </li><li>ピーク張力の時間（ミリ秒）= 最大張力の収縮の発症からの時間</li><li>ハーフ緩和時間（ミリ秒）= ピーク張力のピーク張力の50％までの時間</li><li>強縮張力、P O（MN）= 破傷風中に生成される最大張力</li><li>特定の強縮張力（N / cm 2）と= 強縮張力（MN）/断面積（μm2と ）× 10 5 N / MN•μmの2 / cm 2で </li><li>破傷風の上昇の最大速度（N / S）= 破傷風、すなわち、強縮張力曲線（または、DP O / DT）の最大傾斜でテンション上昇中の緊張の増加の最大速度</li><li>ハーフ緩和の強縮張力（MS）= 刺激の停止からの刺激の終了時に緊張の50％までの時間</li><li>単収縮張力から強縮張力の比、P、T / P O = 最大単収縮張力/最大強縮張力</li><li>疲労指数= 最大等尺性張力の低周波疲労の2分後の張力の比</li></ol> 5。代表的な結果<img src="/files/ftp_upload/2582/2582fig1.jpg" alt="図1" /> 図1。 EDL筋肉の解剖。後肢muscles.TA、前脛骨筋。B、EDLの露出（指伸筋屈筋）筋肉の、露出。C、EDL腱の縫合糸の添付ファイル。D、テンショントランスデューサバス（側面から見た図）。E、EDLはお風呂にマウント（上記から表示）。の筋肉は、不完全に例示目的のためのバッファーに浸漬し、実際には、筋肉が完全に乾燥から防ぐために浸漬する必要があります。 <img src="/files/ftp_upload/2582/2582fig2.jpg" alt="図2" /> 図2。張力曲線の例。最大の単収縮張力（P t）は、収縮時間（CT）とハーフ緩和時間（HRT）を示す単収縮張力曲線の、例。バー、1S。B、最大強縮張力を示す強縮張力曲線（P O）の例とハーフ緩和の強縮張力（HRTT）。バー、1S。 <img src="/files/ftp_upload/2582/2582fig3.jpg" alt="図3" /> 図3。力周波数の関係の解析の例。増分刺激の周波数で生成される、緊張が。B、30MHzの。バー、80ミリ秒。C、140MHzでパルス列の例でパルス列の例。バー、80ミリ秒。D、に示されているデータから得られる力周波数の曲線の例。力周波数の曲線の形状は筋肉の強さの特徴である、と別の動物から筋肉の間で比較することができます。 <img src="/files/ftp_upload/2582/2582fig4.jpg" alt="図4" /> 図4。低周波数の疲労解析の例。 、指示された時点（B、C、D）でのパルス列の低周波stimulation.Examplesの期間に生成された減分の緊張を以下に示します。Eは、に示されているデータから得られる低周波疲労曲線の例。低周波数の疲労曲線の形状は筋肉の強さの特徴である、と別の動物から筋肉の間で比較することができます。

<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>

利害の衝突は宣言されません。

我々は、マウスからの外植後肢筋のミオ - 機械的特性を評価するための詳細なアプローチについて説明します。 EDLは、ために前脛骨筋の後ろに、その後方の位置から解剖することはより困難ながら、その顕著な腱の添付ファイルのために足首と膝の関節に脛骨の前方より評価することが容易です。これらの腱は筋肉のストリップミオグラフで取り付け容易。対照的に、より簡単にアクセスする前脛骨は 、両方の筋肉を損なうことなく解剖し、そしてミオグラフでしっかりとマウントすることは非常に困難に、膝関節で広い、ほとんどatendinous添付ファイルを持っています。我々はまた、急速に生理的緩衝液と温度で酸素浴で筋肉をマウントすることを指摘して筋肉の機械的性質を保持することが不可欠です。我々はこれらの条件の下で筋肉の応答が大幅に変更することなく、最大30分間この分析を繰り返すことができることを見出した。最後に、これが筋機能に悪影響を与える、とミオ機械的な力を過小評価する結果となることができるように筋線維は、解剖や取り付け手順中に触れられないことが不可欠である。これらの手順に従うことにより、この分析は、筋肉の機能6,7,8,9の遺伝的改変の影響だけでなく、筋肉の病気1,2,3,5のマウスモデルにおいて治療的介入との比較を評価するために堅牢な定量的なアプローチを提供。

ソリューションとメディア 乳酸リンゲル液 <ul><li> 100mMのNaCl </li><li> 30mMのCH3CH（OH）COONa（乳酸ナトリウム） </li><li> 4 mMのKClを</li><li> 1mMのCaCl 2 2H 2 O（塩化カルシウム二水和物） <ul><li> 6.75にpHを調整する</li></ul></li></ul> クレブスヘンゼライト溶液 <ul><li> 118 mM NaClを</li><li> 4.7 mMのKClを</li><li> 1.25 mMのCaCl 2を </li><li> 1.2 mMのMgCl 2 </li><li> 1.2 mmのKH 2 PO 4 </li><li> 25mMのNaHCO 3を </li><li> 11 mMグルコース<ul><li> O 2 / CO 2（95％/ 5％）ガスとの平衡によって7.2から7.4にpHを調整する</li></ul></li></ul> ペントバルビタール <ul><li>滅菌水で5 mg / mLの希釈標準溶液</li></ul>

myo-mechanical analysis of isolated skeletal muscle

筋肉の疾患1,2,3の治療のための治療的介入の in vivo効果を評価するために、定量的な方法が必要とされているその治療を受けた筋肉の力発生や易疲労性を測定する。我々は、マウスから新鮮な植後肢筋のミオ - 機械的特性を評価するための詳細なアプローチについて説明します。使用して、、そして最大の痙攣と強直性緊張、収縮時間、およびハーフ緩和時間の測定、我々は、筋肉ストリップミオグラフ（デンマークミオテクノロジーモデル820MS）の筋肉を取り付け、マウス指伸筋の筋筋の非外傷性収穫を記述方形パルス刺激（モデルS48、グラス技術）。これらの測定値を使用して、我々は特定のけいれんや筋肉の断面積で正規化強直性張力の計算、筋間の強縮張力の比、力周波数の関係の曲線と低周波の疲労曲線4を示しています。この分析は、筋肉の病気1,2,3,5だけでなく、筋肉の機能6,7,8,9の遺伝的改変の効果の比較のマウスモデルにおいて治療的介入の間に定量的に比較する方法を提供する。

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

評価する<em&gt; in vivoで</em筋肉の疾患の治療的介入の&gt;効果、方法、治療筋肉の力発生や易疲労性を定量するために必要とされる。我々は詳細植したマウスの後肢筋のミオ - 機械的性質を評価するアプローチを。この分析では、筋機能に関する遺伝子改変の影響だけでなく、筋肉の疾患のマウスモデルにおける治療法の比較を定量に強力なアプローチを提供します。

単離骨格筋のミオ - 機械的分析

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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JoVE Journal

Medicine

26.7K ビュー.  University of California San Francisco. 評価する in vivoで効果、方法、治療筋肉の力発生や易疲労性を定量するために必要とされる。我々は詳細植したマウスの後肢筋のミオ - 機械的性質を評価するアプローチを。この分析では、筋機能に関する遺伝子改変の影響だけでなく、筋肉の疾患のマウスモデルにおける治療法の比較を定量に強力なアプローチを提...

ビデオ: 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.

プロテオミクスから骨格筋の性別二型

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

医学

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;

脛骨神経切断 - マウスにおける脱神経誘発性骨格筋萎縮のために標準化されたモデル

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.

変更されたベルイストローム法を用いたヒト骨格筋生検手順

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.

経皮針生検によって得られた骨格筋組織から単離されたミトコンドリアの調製と呼吸計測評価

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.

骨格筋疾患の定量的磁気共鳴イメージング

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.

マウスの圧負荷モデルを用いた冠動脈の流れと冠血流準備の超音波ベースのアセスメント

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.

組織学的分析のために筋肉凍結切片の調製でのやっていいことといけないことを行います

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.

人間<em&gt;外側広筋</emワイル・ブレークスリーConchotomeを使用&gt;骨格筋生検

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.

ヒトにおける労作骨格筋損傷の誘導および評価

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.

リン-31磁気共鳴分光法：測定するためのツール<em&gt;インビボ</emヒト骨格筋における&gt;ミトコンドリア酸化的リン酸化能力

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