Name: application of chronic stimulation to study contractile activity-induced rat skeletal muscle phenotypic adaptations
Uploaded: 2018-01-25T13:00:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations at JoVE.com

原稿の彼の専門の読書のリアム フォートタイロンに感謝しております。この作品は、D. A. フードに自然科学と工学研究審議会のカナダ (レベル) からの資金によって支えられました。D. また、A. フード、細胞生理学のカナダの研究の椅子の保持者です。

すべての動物関連のプロシージャはレビューされ、ニューヨーク大学動物ケア委員会によって承認されました。ヨーク大学の動物実験施設に到着すると、すべてのラットは食品提供広告自由で、手術前に自分の環境に順応する 5 日間の最小値を与えられました。他種15,17,22にこのプロトコルが以前適用されている現在の紙 Pette と同僚の23の先駆的な仕事の上に構築、特にラット モデルに焦点を当ています。
1. 慢性収縮活動ユニットの準備
<ol><li>電圧計を使用して、コイン リチウム電池の可能性を確認してください。 注: 各電池の可能性は、3.0 ± 0.10 V をする必要があります。</li>
	<li>合計可能性が 6-9 V、ユニットのスロットに 2 つまたは 3 つの電池を挿入します。 注: この実験中 (6 または 9 V) 維持するためにどのように多くの潜在性を考慮する研究者の裁量次第です。研究デザイン、刺激の目的の強度に応じて 2 または 3 電池を使用できます。</li>
	<li>ユニットがきちんと働いているパルス指標経由ポータブル赤外線ストロボの光によって放射される 1 のパルス ユニットを公開することによってを確認します。</li>
</ol>2. 慢性の収縮活動の手術
注: は、手術前にすべての手術器具を滅菌します。中および手術後すぐに、ラットの体温は、加熱パッドによって維持されます。手術用覆布に手術の手順を実行することをお勧めします。外科医は、滅菌手術用手袋として清潔な白衣を着用ください。必要な場合は、使い捨ての防毒マスクを着用することをお勧めします。
<ol><li>麻酔ラット 1 3% 未満イソフルラン吸入酸素は、ガスの気化器システムが運営しております。後肢のつま先ピンチをチェックすることによって、呼吸の深さそして率を観察することによって、動物が鎮静完全に確認してください。乾燥を避けるために目に眼の潤滑剤を適用します。メロキシカム (0.5 mg/mL) の 2 mg/kg の皮下注射を適用します。 注: またお勧め中や手術後の痛みを最小限に抑えるため鎮痛 (例えば、メロキシカム プラス リドカイン) のマルチ モーダル アプリケーションを持っています。</li>
	<li>優しく剃る、首の後ろから胴体周りのストリップと同様、左後肢周りの後ろに前肢の前胸部全体。ヨードとアルコール消毒する剃毛エリアを軽く拭きます。</li>
	<li>動物の胃の上に敷設、剃毛地域 (肩甲骨の間明白な領域) の中央に首の後ろに小さな切開 (~0.5 cm) を作るメス (第 10 刃) を使用します。</li>
	<li>一般的な腓骨神経を探します。<ol>
<li>その右側に動物をロールし、2 〜 3 cm-長い左後肢の皮膚にカットします。膝関節のディンプルと尾の原点の近くに戻ると landmarked が大腿筋群周囲切開領域をターゲットします。 注: 本体の位置を変更するとき最初の切開領域を汚さないように気をつけてください。</li>
		<li>外科はさみを湾曲した鈍い先端を使用して、皮下のエリアを広く分析 ~3.5 - 4 cm、開いた肌と基になる筋肉の間のポケットを作るために基になる筋肉から皮膚を分離します。全周切開 (~1.5 cm2) の基になる組織から皮膚を開きます。</li>
		<li>外科はさみ、はさみの先端が筋肉を介して直接ダウン切っている確保と大腿二頭筋筋小切開 (0.5 cm 未満) を作る。</li>
		<li>内部の筋肉と、総腓骨神経が表示されるまで優しくカット領域を開く (外部の筋肉組織 (すなわち、大腿二頭筋) の深さの ~0.5 cm あたりです)。鉗子を使用して、優しくタッチ/目に見える神経とピンチ ターゲット筋群 (例えばTA 筋) とつま先 (目に見える屈)、総腓骨神経が分離されたことを確認するための応答を観察します。 注: この手順は、切断または神経の損傷を避けるために細心の注意されるべきであります。</li>
		<li>ウィンドウのサイズがウィンドウの中央に横たわっている腓骨神経と ~1.5 cm2金属リトラクターで開く引いてウィンドウを修正します。ストラップやテーブルの表面 (または手術ボード) に留まり、ゴムバンドに接続されている小さな金属製のフックを使用して (図 2 a)。</li>
	</ol></li>
	<li>神経の両側にワイヤーを取り付けます。<ol>
<li>ポリテトラフルオロ エチレン (PTFE) の 50 〜 60 cm を準備-ステンレス鋼細線をコーティングし、半分に折る。 注: 手術前に紫外線の下で PTFA めっきなしワイヤを公開すると便利場合があります。</li>
		<li>30 cm ステンレス鋼棒のスリットにワイヤーの折れ曲がった部分をフックします。足と背中の中心に沿って L 字型パターンで一緒に、首の後ろの小さな切開領域に向かって後肢のオープン ポケットから皮下、ワイヤー、ロッドを渡します。</li>
		<li>後肢でワイヤの 2 つの端を見つけます。すべてのワイヤ PTFE 絶縁電線をストリップします。~1.5 cm ワイヤーの両端を取り除くメスを使用して、慎重に。ワイヤーは擦り切れた様になって、それらをカットし、再ストリップします。(5 回)、鈍化 21 G 針の周り裸ワイヤの両端をラップ コイルを作るします。コイルが正しく行われると、そこから針をリリースします。</li>
		<li>サイズ 6-0 外科シルクを使用して、それぞれ、総腓骨神経 (図 2 a) のどちら側にコイルの固定します。<ol>
<li>コイルの非常に端に結び目を作るし、左側にある神経の縫合します。コイルが、神経から 1.5 〜 2.5 mm であることを確認します。</li>
			<li>コイルを固定するには、コイルに沿って 2 つまたは 3 つの追加縫合を適用します。</li>
			<li>神経の右側にこれらの手順を繰り返します。</li>
		</ol>
</li>
		<li>抗生物質溶液 (生理食塩水でアンピシリン; 132 mg/mL) の 2 〜 3 滴を適用し、サイズ 5-0 絹糸を使用してウィンドウ (つまり、大腿二頭筋筋肉組織) を慎重に縫合します。</li>
	</ol></li>
	<li>(約人差し指の直径) 線・ (ヒップ) 上記約大腿二頭筋筋肉の切開部を縫合上皮下ポケットにプッシュの残りの余裕が緩く風します。</li>
	<li>抗生物質溶液再び (生理食塩水でアンピシリン; 132 mg/mL) 2 〜 3 滴を適用します。ホチキス止めして開かれた皮膚を閉じます。</li>
	<li>(首の切開部から出てくる) ワイヤーを CCA 刺激装置に接続します。<ol>
<li>ワイヤーとピンのソケットに接続します。<ol>
<li>切開 2 ワイヤー エンド (腓骨神経のいずれかの側に縫合コイルにこれらの鉛) を作成する首の上部に出てくるワイヤー ループをカットします。</li>
			<li>メスで ~0.5 cm カット擦り切れているワイヤーをワイヤーの端を離れてストリップを使用してください。</li>
			<li>ゆっくりピン ソケットの穴にワイヤの裸の部分を押すし、ピンのソケットの電線をはんだ付け、はんだごてを使用して。</li>
		</ol>
</li>
		<li>必要に応じて、線の接続を確認します。<ol>
<li>ワニ口クリップを介して大規模なベンチトップ型刺激装置ユニットにピンを接続します。</li>
			<li>単一のパルス 9 V (0.1 ms、10 Hz) TA 筋契約および左の足の dorsiflexes を確認するを提供します。</li>
		</ol>
</li>
		<li>〜 4 cm × 4 cm は、滅菌ガーゼのパッドを介してピン接続ワイヤの両端を渡します。</li>
		<li>ピンを CCA の刺激装置に接続します。<ol>
<li>刺激装置ボックスのベースの穴に通してワイヤーを渡します。 注: このボックスは、CCA の刺激装置の自家製チャンバーは 3.5 cm × 3.5 cm × 2.5 cm13。</li>
			<li>CCA ユニットの接続ソケットにピンを挿入します。CCA ユニットを商工会議所にそっと入れます。粘着タックを使用して、チャンバーの下部に CCA ユニットに固定します。</li>
		</ol>
</li>
		<li>アスレチック テープまたは多孔質のサージカル テープを使用して、坊主の胴体周りのテープでチャンバーを固定します。テーピングの 3 つの層の部屋の上部を閉じます、ボックス (図 2 b) をセキュリティで保護する刺激装置ボックスの側面にテープをラップすることによって完了します。</li>
	</ol></li>
	<li>CCA が赤外光の単一パルス ユニットを公開することによって動作しているを確認 (波長スペクトル > 770 nm) ポータブル赤外線ストロボの光から放出します。 注: CCA が正しく動作している場合、研究者が後肢筋 (すなわちTA) の近赤外光応答の契約先を見ることができるします。</li>
	<li>ラットを観察し、それは完全に意識を取り戻したまでその温度を監視します。他の動物からの害を防ぐために単一乗員ケージの家および刺激装置の損傷や動物への損傷のリスクを軽減する研究の残りの部分の檻の中、トンネル、またはプラスチック製のオブジェクトを放置しないでください。アモキシシリン含まれて水ボトル (0.5 mg/mL) を提供します。</li>
	<li>次の手術は、少なくとも 72 時間続くすべての 24 h 皮下メロキシカムの 1 mg/kg の投与量を適用します。</li>
</ol>3. 慢性の収縮活動
<ol><li>手術後切開と縫合周辺で/骨格筋の完全復旧のため、少なくとも 5-7 日を許可します。 注: 中と CCA の手順後、(例えば、食べて、飲んで、や移動) 彼らの行動を観察することによってそれぞれの動物の状態を徹底的にチェックします。さらに、CCA の手順の前後に体重の変化をチェックすることによって、重度のストレスや有害効果を決定します。</li>
	<li>赤外光の単一パルス刺激装置ユニットを公開することによって CCA をオンに CCA 刺激の日 (波長範囲 > 770 nm) ポータブル赤外線ストロボの光で。</li>
	<li>10 Hz CCA 刺激の 3 または 6 h を適用します。 注: 刺激のための時間枠は、研究者までです。これらの実験で刺激の周波数は変更されて決して、私たちの経験では、6 h/日に 3 からの刺激を拡張することによってミトコンドリアの適応にのみ非常に控えめな拡張が観察されています。30-60 分毎、刺激と動物を確認可能であれば。</li>
	<li>目的 CCA 期間が過ぎると、赤外光照射 (ユニットの電源を入れると同じプロシージャ) を介して CCA ユニットの電源を切ります。</li>
	<li>複数の日を適用する場合は、3.2 の手順を繰り返します。3.4 を.</li>
	<li>組織の採取のタイミングを決定します。たとえば、cca (すなわち、6 h の最後の CCA 刺激後 18 h)、CCA の手術の手順の間に実行される、麻酔下で行われる最後の試合の発症後 24 時間の組織を収集します。すべての組織を収集、直後後、中動物は麻酔に心臓を切除動物を安楽死します。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronology+of+UPR+activation+in+skeletal+muscle+adaptations+to+chronic+contractile+activity.">Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity.</a> Am J Physiol Cell Physiol. 310 (11), C1024-C1036 (2016).</li><li>Ljubicic, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+an+attenuated+mitochondrial+adaptive+plasticity+in+aged+skeletal+muscle.">Molecular basis for an attenuated mitochondrial adaptive plasticity in aged skeletal muscle.</a> Aging (Albany NY). 1 (9), 818-830 (2009).</li><li>Schwarz, G., Leisner, E., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+telestimulation+systems+for+chronic+indirect+muscle+stimulation+in+caged+rabbits+and+mice.">Two telestimulation systems for chronic indirect muscle stimulation in caged rabbits and mice.</a> Pflugers Arch. 398 (2), 130-133 (1983).</li><li>Simoneau, J. A., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+effects+of+chronic+nerve+stimulation+upon+tibialis+anterior+muscle+in+mouse,+rat,+guinea+pig,+and+rabbit.">Species-specific effects of chronic nerve stimulation upon tibialis anterior muscle in mouse, rat, guinea pig, and rabbit.</a> Pflugers Arch. 412 (1-2), 86-92 (1988).</li><li>Ohlendieck, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+low-frequency+stimulation+on+Ca2+-regulatory+membrane+proteins+in+rabbit+fast+muscle.">Effects of chronic low-frequency stimulation on Ca2+-regulatory membrane proteins in rabbit fast muscle.</a> Pflugers Arch. 438 (5), 700-708 (1999).</li><li>Brown, M. D., Cotter, M. A., Hudlicka, O., Vrbova, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+different+patterns+of+muscle+activity+on+capillary+density,+mechanical+properties+and+structure+of+slow+and+fast+rabbit+muscles.">The effects of different patterns of muscle activity on capillary density, mechanical properties and structure of slow and fast rabbit muscles.</a> Pflugers Arch. 361 (3), 241-250 (1976).</li><li>Skorjanc, D., Traub, I., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identical+responses+of+fast+muscle+to+sustained+activity+by+low-frequency+stimulation+in+young+and+aging+rats.">Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats.</a> J Appl Physiol. 85 (2), 437-441 (1998).</li><li>Kim, Y., Triolo, M., Hood, 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=Impact+of+Aging+and+Exercise+on+Mitochondrial+Quality+Control+in+Skeletal+Muscle.">Impact of Aging and Exercise on Mitochondrial Quality Control in Skeletal Muscle.</a> Oxid Med Cell Longev. 2017, 3165396 (2017).</li><li>Callewaert, L., Puers, B., Sansen, W., Jarvis, J. C., Salmons, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+implantable+device+for+investigating+the+adaptive+response+of+skeletal+muscle+to+chronic+electrical+stimulation.">Programmable implantable device for investigating the adaptive response of skeletal muscle to chronic electrical stimulation.</a> Med Biol Eng Comput. 29 (5), 548-553 (1991).</li><li>Kern, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+counteracts+muscle+decline+in+seniors.">Electrical stimulation counteracts muscle decline in seniors.</a> Front Aging Neurosci. 6, 189 (2014).</li><li>Zampieri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+exercise+in+aging+human+skeletal+muscle+increases+mitochondrial+calcium+uniporter+expression+levels+and+affects+mitochondria+dynamics.">Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics.</a> Physiol Rep. 4 (24), (2016).</li></ol>

著者が明らかに何もありません。

運動、低周波筋刺激を通じて生体内の慢性的な収縮活動 (CCA) モデルは13,24,25を行使する筋肉表現型適応を研究するためのエクセレント モデル,26. 以前研究20,27に示すように、CCA は、研究者がトレーニング ボリュームと周波数 (すなわち時?...

骨格筋は、その生体エネルギーと物理構造1の変化を通じてトレーニングに適応できます。持久性トレーニングによってもたらされる主な変更の 1 つはミトコンドリア生合成、ミトコンドリア コンポーネント (例えばチトクローム c 酸化酵素 [シクロオキシゲナーゼ] サブユニット) 発現の増加によって評価することができますの式転写コアクチベーター、pgc-1 α2。研究の増加は、ミトコンドリア代謝と mitophagy を含む、他の多くの要因は筋肉の適応の重要なも示されています。ただし、急性または慢性的な運動によって機構がこれらを調節する骨格筋内のプロセスはまだ明確ではないです。
運動誘発性筋適応を規制する経路を記述するには、様々 な運動モデルはトレッドミル、ホイールを実行して、水泳運動を含む、齧歯類での試験でよく使用されています。ただし、これらのプロトコルは 4 〜 12 週間はこれらの表現型の変化3,4、5の観察に必要な制約があります。実験方法として低周波刺激誘発性慢性収縮活動 (CCA) 効果的として使用されている、それは大幅に短い期間で筋肉の適応をもたらす (すなわち、 7 日前まで) その効果と匹敵する、または他の運動のプロトコルよりもあります。さらに、ホルモン6温度7、および神経学的な効果8の存在は、慢性運動に対する筋特異応答を理解することは困難することができるのです。たとえば、甲状腺ホルモン9,10インスリン様成長因子 (IGF)-111は、トレーニングによる筋肉の適応、また骨格その他のシグナル伝達経路を調節することがありますを仲介に同定されています。筋肉。特に、CCA 誘発効果は、全身的要因、骨格筋の収縮活性への直接反応に配置するフォーカスを許可によって規制されて最小限。
CCA の外部ユニットはタイラーとライト12、初し、変更12で開発されています。一言で言えば、単位は 3 つの主要部分から成る: パルス インジケーター (図 1)、パルス発生器、赤外光への暴露によってオン/オフにすることができます赤外線検出器。刺激装置の詳細な回路設計がされている13で説明しました。CCA の詳細と特定の機能は、大きい数のレビューの深さの記事14,15,16,17。低周波で、総腓骨神経をアクティブにする刺激プロトコルを設計する簡単に言えば、(すなわち、 10 Hz)、神経筋肉 (前脛骨筋前部 [TA] および [EDL] 長趾伸筋) が契約を締結させると、所定の時間 (例えば、3 〜 6 時間) の長さ。時間をかけて、これは毛細血管密度18とミトコンドリア コンテンツ19,20,21の増加によって示されるより好気性表現型に前述の筋肉をシフトします。したがって、このメソッドは、いくつかのラット骨格筋内の主要な持久力トレーニング適応を模倣する実績のあるモデルです。
研究者は、彼らの運動の研究で、このモデルを適用できますので、CCA を誘発する電極注入手術の詳細な手順について述べる。CCA は、したがって運動トレーニングの開始に続く両方の序盤とそれ以降の時間ポイントで様々 な分子とシグナル伝達イベントの調査のための効果的なツールを提供する筋適応の時間的経過を研究するための優れたモデルです。

<table><tbody><tr><td>Sprague Dawley Rat</td><td>Charles River</td><td>Strain 400</td><td></td></tr><tr><td>Chronic contractile activity unit</td><td>Home-made</td><td>n/a</td><td></td></tr><tr><td>CCA unit protective box (3.5 x 3.5 x 2.5 cm)</td><td>Home-made</td><td>n/a</td><td>Box should be made of opaque material or covered in an opague tape</td></tr><tr><td>Coin lithium ion batteries (3V)</td><td>Panasonic</td><td>CR2016</td><td></td></tr><tr><td>Medwire</td><td>Leico Industries</td><td>316SS7/44T</td><td></td></tr><tr><td>Solder pin (socket)</td><td>Digi-Key</td><td>ED6218-ND</td><td></td></tr><tr><td>Zonas porous tape</td><td>Johnson &amp;amp; Johnson</td><td>5104</td><td></td></tr><tr><td>Suture silk (Size 5)</td><td>Ethicon</td><td>640G</td><td></td></tr><tr><td>Suture silk (Size 6)</td><td>Ethicon</td><td>706G</td><td></td></tr><tr><td>Curved blunt scissor (11.5 cm Length)</td><td>F.S.T.</td><td>14075-11</td><td></td></tr><tr><td>Curved blunt scissor (15 cm Length)</td><td>F.S.T.</td><td>14111-15</td><td></td></tr><tr><td>Delicate haemostatic forceps (16 cm Length)</td><td>Lawton</td><td>06-0230</td><td></td></tr><tr><td>Scalpel</td><td>Feather</td><td>3</td><td></td></tr><tr><td>Curved forceps</td><td>F.S.T.</td><td>11052-10</td><td></td></tr><tr><td>Stainless-steel rod (30 cm; 7mm diameter)</td><td>Home-made</td><td>n/a</td><td>Rod should have 5 mm slit in one end to hold the wire for tunneling under the skin</td></tr><tr><td>Clip applying forceps</td><td>KLS Martin</td><td>20-916-12</td><td></td></tr><tr><td>Staples (clips)</td><td>Bbraun</td><td>BN507R</td><td></td></tr><tr><td>Metal hooks/retractor</td><td>Home-made</td><td>n/a</td><td></td></tr><tr><td>Povidone-iodine (500 mL)</td><td>Rougier</td><td>#NPN00172944</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Novopharm</td><td>#DIN00872644</td><td></td></tr><tr><td>Metacam</td><td>Boehringer</td><td>#DIN02240463</td><td></td></tr><tr><td>Digital multimeter (voltmeter)</td><td>Soar Corporation</td><td>ME-501</td><td></td></tr><tr><td>LED digital stroboscope</td><td>Lutron Electronic Enterprise</td><td>DT-2269</td><td></td></tr></tbody></table>

application of chronic stimulation to study contractile activity-induced rat skeletal muscle phenotypic adaptations

骨格筋は、その生化学的・生理学的特性が大幅に慢性運動に応えて変更適応性の高い組織です。筋肉の様々 な適応をもたらすメカニズムを調べるためには、トレッドミル、ホイール ランニング、水泳など運動プロトコルの数は、動物実験で使用されています。ただし、これら運動のモデル特定の筋肉収縮による適応の研究に応用が少なくて、体液や神経の要因によっても規制が筋の適応を達成するために時間の長い期間を必要とします。それは正常に全身的要因から独立して、7 日以内、筋肉ミトコンドリア適応につながる可能性としての慢性的な収縮活動 (CCA) を誘発する間接低周波刺激 (10 Hz) は運動トレーニングのための代替モデルとして使用されています。広範なアプリケーションを将来的に研究、ラットの骨格筋に CCA の治療を適用する必要な手術手技の詳細を説明します。

我々 は、慢性的な収縮活動 (CCA) が骨格筋内有利なミトコンドリアの適応を誘発する効果的なツールであることを示しています。CCA (6 h/日) の 7 日間を受けるラットは、非刺激対側 (コントロール) 後肢と比べて刺激を受けた筋肉のミトコンドリア生合成を拡張を表示します。ミトコンドリア生合成の増加が他のキーのミトコンドリア蛋白質コックスの標高とともに、?...

Watch this Scientific Journal Video about Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations at JoVE.com

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

このプロトコルでは、ラット後肢筋の刺激による骨格筋適応を観察する運動の慢性収縮活動モデルの使用について説明します。

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

application-chronic-stimulation-to-study-contractile-activity-induced

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.7K ビュー.  York University. このプロトコルでは、ラット後肢筋の刺激による骨格筋適応を観察する運動の慢性収縮活動モデルの使用について説明します。

ビデオ: Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Myo-mechanical Analysis of Isolated Skeletal Muscle

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 generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

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 ...

医学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

アン in vivoで</em収縮で誘起された損傷と回復の非侵襲的モニタリングの>齧歯類モデル

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

携帯収縮機能を評価するために、マイクロスケールのシリコンカンチレバーの活用<em&gt;体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

生物工学

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

の応用<em&gt;インビボ</em組織工学骨格筋の修復を評価するためのラット前脛骨の&gt;機能テスト

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

電気的活動の変化を受けた個々の生きたゼブラフィッシュの骨格筋成長のリアルタイムおよび反復測定

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

発生生物学

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

ラットのための手順<em&gt;その場で</em&gt;骨格筋収縮特性

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

生物学

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

ヒト骨格筋微小組織における骨格筋の健康の機能指標の評価

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

隔離された骨格筋の収縮、倦怠感や交互の ex vivo での評価

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

ヒト神経筋接合部の光遺伝学的モデルの工学と特徴付け

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

マウスの心臓変力作用の調​​査のために分離された乳頭筋の収縮の測定

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

要約

さらに動画を探す

この動画の章

単離骨格筋のミオ - 機械的分析

アン in vivoで齧歯類モデル

携帯収縮機能を評価するために、マイクロスケールのシリコンカンチレバーの活用

の応用

電気的活動の変化を受けた個々の生きたゼブラフィッシュの骨格筋成長のリアルタイムおよび反復測定

ラットのための手順

ヒト骨格筋微小組織における骨格筋の健康の機能指標の評価

隔離された骨格筋の収縮、倦怠感や交互の ex vivo での評価

ヒト神経筋接合部の光遺伝学的モデルの工学と特徴付け

マウスの心臓変力作用の調査のために分離された乳頭筋の収縮の測定

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

要約

さらに動画を探す

この動画の章

単離骨格筋のミオ - 機械的分析

アン in vivoで齧歯類モデル

携帯収縮機能を評価するために、マイクロスケールのシリコンカンチレバーの活用

の応用

電気的活動の変化を受けた個々の生きたゼブラフィッシュの骨格筋成長のリアルタイムおよび反復測定

ラットのための手順

ヒト骨格筋微小組織における骨格筋の健康の機能指標の評価

隔離された骨格筋の収縮、倦怠感や交互の ex vivo での評価

ヒト神経筋接合部の光遺伝学的モデルの工学と特徴付け

マウスの心臓変力作用の調​​査のために分離された乳頭筋の収縮の測定

マウスの心臓変力作用の調査のために分離された乳頭筋の収縮の測定