この作品はBrotto Mに趙X、​​RO1-AR061385にMA、JとGOグラントRC2AR05896にAHA SDG 10SDG2630086によってサポートされていました

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収縮力と疲労の測定は、骨格筋の機能を総合的に評価するために重要である。このアッセイの主要な目的は、サルコペニアや筋肉疲労などの特定の病理学的な条件の下での筋肉の力の変化と疲労特性を明らかにし、筋収縮に薬/試薬の効果をテストすることです。筋力が密接に細胞内Ca 2 +の放出と関連しているので、細胞外Ca 2 +流入とこれらの2つの間のクロストークは、我々?...

骨格筋は、骨格の骨に付着し、中枢神経系の制御の下で収縮力を発生させる。興奮収縮連関（ECC）は機械的な応答に電気刺激に変換するプロセスを指します。 Ca 2 +シグナル伝達は骨格筋における収縮機能の重要なコンポーネントです。効果的なのCa 2は筋小胞体（SR）から+動員は重要な筋肉細胞1、2、ECCのための成分と、細胞内Ca 2 +シグナル伝達の変化は、筋疾患3-5の番号に対応する収縮機能障害の根底にある。筋収縮性の適切な評価は、Ca 2に不可欠と無料でご利用いただけます+イメージングおよび他のアッセイは、収縮性のレベルではなく、運動レベルではないだけで、骨格筋の機能についての洞察を得るために。力と速度はまたの重要な特性を知らせるために得ることができる筋力と異なる生理学的および病態生理学的条件下でECC処理のステータス。 この研究分野は非常に肥沃な豊かな歴史と筋収縮の多くの理論は、二千6の上に現れました。近代的な筋肉の研究はおそらくレーウェンフック6による筋線維のクロス線条と筋原繊維の顕微鏡観察で1674年から1682年に始まる。その神経が遠い電気機械7月9日から火花放電時にメスで触れたときの、ほぼ一世紀後、ルイジ·ガルバーニはカエルの筋肉の収縮を激しくすることを観察した。収縮はまた、金属導体を介して筋肉に脚の神経を接続することによって生成することができる。ガルバーニが提唱する複雑な電気シグナル伝達機構の詳細は、最終的には電気生理学の基礎となった彼らの有名な方程式10,11にホジキン、HuxleyとKatzによって策定されました。凛の驚くべき観察カエルの心臓や骨格筋12月15日の収縮に細胞外Ca 2 +の影響についてドイツは筋収縮性16、17の重要な調節因子としてのCa 2 +の認識の最初の大きな一歩を表しています。 1980年代から現在に至るまで筋収縮フィールドで発見のバーストが原因で筋肉の収縮およびマウス骨格筋18で疲労プロトコルの導入を実現しました。ジョーンズとエドワーズは、低周波断続的疲労（力の運動誘発還元）19は ECCの機械ではなく、収縮装置の変化は認められなかった。示唆する最初でした一方、1980年代後半と1990年代前半に、Kolkeck ら 20、ベックとノセク21、リード22は 、骨格筋の収縮にテオフィリン、cortiosterone、およびフリーラジカルの影響を研究する齧歯類モデルから横隔膜筋を使用していたブルックスとFaulknerがマウス22から高速と低速の筋肉の力が繰り返されると、パワー測定の測定結果を報告する最初でした。また、Lannegren、Westerblad、子羊、そしてWesterbladは直接細胞内Ca 2 +調節にex vivoでの収縮をリンクした最初と筋肉疲労23,24におけるアシドーシスの役割に疑問を始めました。 私たちの研究所では、大幅に細胞内Ca 2 +の監視で、そのままマウスの筋収縮の研究の組み合わせを使用することによって2000年代初頭に新規遺伝子の理解に向けて調節し、筋肉の収縮、疲労、老化において重要な役割を持つ筋肉のECCに関する規制の役割を持つので、貢献してきました無傷の肌の筋線維と分子遺伝子操作3-5、25から29。 ここでは、マウス孤立ヒラメ筋と長趾伸筋（EDの収縮を測定するための実験プロトコルを詳述L）が異なる収縮特性を主に遅い酸化（タイプIおよびIIa筋線維）と、主に高速glyocolytic筋（IIb型とIIxの筋線維）に対応して筋肉、。このプロトコルでは、無傷の筋腱複合体を単離し、純酸素または酸素（95％）と、CO 2（5％）の混合物のいずれかに付属して、ADI PowerLabのRadnottiチャンバーシステムでびっしょり。収縮力は、草の刺激からの電気刺激によって生成され、マクロのルーチンのカスタマイズは、データの取得、収集、デジタル化、およびストレージを制御することができ、アナログ·デバイセズPowerLab/400システムと統合された力変換器を用いて検出した。このセットアップでは、筋力、筋パワーだけでなく、力対周波数の関係、筋肉疲労、筋肉疲労、スピード、筋収縮の全体的な速度論的性質からの回復を測定することができます。また、筋収縮に対する薬物の効果は、これらの実験を通して監視することができます。 </p&gt; この方法の利点は、筋肉を収縮させるの固有の特性を直接評価できるように、骨格筋から離れて神経や血管成分を除去することにあった。また、ex vivoでの収縮性アッセイは、骨格筋の機能のための彼らの生理的役割を定義するために、様々なイオン透過チャネルやトランスポーターの薬理操作の使用を可能に孤立筋肉を取り囲む細胞外環境の操作を可能にする。 このex vivoのシステムは、私たちが最近変更された細胞内Ca 2 +ハンドリング性4に結合されていた特定の変異体の筋肉の準備、の明確なalternan動作を発見することができました。交互脈は疲労プロファイルの衰退期の間に収縮力の変動バーストエピソードとして定義されています。これらのイベントの間に収縮力が瞬間的に力Dのその前のレベルを超えて増加多くのCa 2 +がリリースされているまたは収縮機械がCa 2 +を30に敏感になっているのどちらかが原因と考えられるため、辛い刺激をuring。その交互脈は直接に関係している示唆リアノジンチャネル（RyR）を繰り返し疲労刺激のアゴニストは、すべての機械的交互脈を誘発することができるシクロピアゾン酸（CPA）は、筋小-小胞体カルシウムATPアーゼ（SERCA）の可逆的遮断薬、カフェインの治療4、 ECの結合過程の変調。 in vitroでの収縮のセットアップでのメカニックの交代を誘導し、記録する方法のデモンストレーションは、個々の研究の関心に基づいて、このシステムまたは類似のものを得ることができた多様な実験パラメータを表示する例となっています。 この方法は、筋肉生理学を勉強し研究者に興味があるかもしれません。同様のセットアップは、他から隔離された骨格muscle-tendon/ligament複合体にも使用できます解剖学的部位と同様、単繊維や筋肉のストリップのため。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> 2-APB </td><td> Tocris </td><td> 1224 </td><td> Ca 2 +の数+ SOCおよびTRPなどを含むエントリチャネルのブロッカー</td></tr><tr><td> SKF96365 </td><td>シグマ</td><td> SKF-96365 </td><td> Ca 2 +の数+ SOCと受容体媒介性のCa 2 +流入等を含むエントリチャネルのブロッカー</td></tr><tr><td> BTP-2 </td><td>ミリポア</td><td> 203890-5mgの</td><td>比較的特異的なSOCのブロッカー</td></tr><tr><td> CPA </td><td>シグマ</td><td> C1530 </td><td>リバーシブルSERCAブロッカー</td></tr><tr><td>カフェイン</td><td>シグマ</td><td> C0750 </td><td>高速アクションRyRアゴニスト</td></tr><tr><td> Radnoti 4つの単位ティッシュオーガンバスシステム</td><td> Radnoti </td><td> 159920 </td><td></td></tr><tr><td>組み合わせティッシュサポート/刺激電極</td><td> Radnoti </td><td> 160151 </td><td>縦のジグザグは、組織のサポートをタイプジグザグ</td></tr><tr><td>クワッドブリッジアンプ</td><td> ADInstruments </td><td> FE224 </td><td></td></tr><tr><td> PowerLab/400 </td><td> ADInstruments </td><td></td><td>本製品は使用できなくなります。データ·アクイジション·システムの他のバージョンを選択します。 </td></tr><tr><td>力変換器（5ミリグラム - 25グラム） </td><td> ADInstruments </td><td> MLT0201/RAD </td><td></td></tr><tr><td>チャート4.02 </td><td> ADInstruments </TD&gt; <td></td><td> LabChart 7.3 Chartソフトウェアの最新バージョンです。 </td></tr><tr><td> S8800デュアルパルスデジタルスティミュレータ</td><td> GRASSのテクノロジー</td><td></td><td>本製品は使用できなくなります。 S88Xデュアル出力方形パルス刺激器は新しい刺激である。 </td></tr><tr><td> RFトランスのアイソレーションユニット</td><td> GRASSのテクノロジー</td><td>モデルSIU5 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles

ここで説明するには、隔離された骨格筋の収縮を測定する方法である。そのような筋力、筋パワー、収縮動態、易疲労感、疲労後の回復などのパラメータは、そのような興奮性として興奮収縮連関の特定の側面（ECC）のプロセスは、収縮機構とCa 2 +ハンドリング能力を評価するために得ることができます。この方法では、神経と血液供給を削除し、単離骨格筋自体に焦点を当てています。我々は日常的に変調のCa 2 +シグナル伝達経路のに骨格筋の収縮特性を変更する遺伝的要素を識別するためにこのメソッドを使用します。ここでは、in vitroでの筋収縮性アッセイで使用して得ることができる様々な、豊かな情報の一例として新たに同定された骨格筋の表現型、 すなわち 、機械的交互脈を記載している。単一細胞アッセイ、遺伝的アプローチと生化学とこのアッセイの組み合わせstryアッセイは、骨格筋のECCのメカニズムに重要な洞察を提供することができます。

Watch this Scientific Journal Video about Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles at JoVE.com

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

我々は、直接筋力、筋パワー、収縮動態との単離された骨格筋の疲労を測定する方法を説明します<em&gt; in vitroで</em&gt;電場刺激を用いたシステム。カルシウムについての貴重な情報<sup&gt; 2 +</sup&gt;ハンドリング性や筋肉の収縮機械が異なる刺激プロトコルを使用して取得できます。

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, ...

ex-vivo-assessment-contractility-fatigability-alternans-isolated

Research

JoVE Journal

Biology

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

23.6K ビュー.  UMDNJ-Robert Wood Johnson Medical School. 我々は、直接筋力、筋パワー、収縮動態との単離された骨格筋の疲労を測定する方法を説明します in vitroで電場刺激を用いたシステム。カルシウムについての貴重な情報 2 +ハンドリング性や筋肉の収縮機械が異なる刺激プロトコルを使用して取得できます。

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

生物学

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用して哺乳類の骨格筋のDNAトランスフェクションインビボでのエレクトロポレーション

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

ザ ex vivoで</em血管反応性の調査のために>隔離された骨格微小血管の準備

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

筋ジストロフィーのマウスモデルから分離された骨格筋の等尺性と偏心力発生アセスメント

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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 ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

マウスから単離およびインキュベートされた成熟骨格筋におけるインスリンおよび収縮刺激グルコース取り込みの測定

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

成人筋幹細胞機能評価のための単一筋繊維培養アッセイ Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...