この作品は、ドイツ学術協会（KFO 196」Signaltransduktion BEI adaptativenウントmaladaptiven kardialenかいそう-Prozessen」、FR 1638 / 1-2）でと心血管研究DZHK（ドイツ語センター、医療研究のドイツ語センターの一部でサポートされていました、BMBF（教育研究のドイツ省）構想）です。

注：単離手順の基本的なステップは、図1に示されているすべてのステップは、以下のプロトコルに詳細に記載されています。乳頭筋の分離、器官槽チャンバ内に取り付け、取得および分析は、連続し、強制タイムスケールで行われます。 全ての動物実験は、動物の保護に関するドイツの法律に従って行われ、ハイデルベルク大学の倫理審査委員会によって承認されました。 計装の調製<ol><li>収縮の測定ではmultichambered器官槽の設定を使用します。 エキソビボ収縮器官浴のセットアップの構成要素は、 図2に示されています。 注：乳頭筋の準備と実験プロトコルの開始との間の時間、セットアップ機器を最小限に抑え、実験tissue.Thisを集めるの提供しにバッファを前に準備するために組織は、実験を行うために生存し続ける時間の最大量をエス。 </li><li>コンピュータとデータ収集機器を起動します。臓器風呂熱（32°Cまでのプリセット）をオンにし、設定温度にユニットを可能にします。 </li><li>収集装置を準備します。ソフトウェアのマニュアルに記載されたように、データの記録を開始。 </li><li>チェックして、各チャンネルに（必要な場合）、キャリブレーションを実施し、認定重量により供給された2-Gの力に（そのチャネルに関連付けられている）は、それぞれの力変換器から供給される電気信号を標準化するために、すべてのチャネルをゼロに。 </li><li>器官槽室へのガス供給（95％O 2/5％CO 2）をオンにします。連続プロトコル全体の測定中に全てのチャンバをガス。 </li></ol>緩衝液および生理的溶液の調製<ol><li> 119 mMの塩化ナトリウムの最終濃度を達成するクレブス - ヘンゼライト緩衝液を調製します。 25mMの炭酸水素ナトリウム; 4.6のKCl; 11mmのグルコース; 1 mMののNa-Pyruvate; 1.5 mMのCaCl 2を、 1.64 mMのは、4硫酸マグネシウム; 1.18 mMのKH 2 PO 4、（ 表1生理溶液を参照）pHを7.4に調整します。 </li><li>追加の30mMの2,3-ブタンジオンモノオキシム（BDM）と50 IEヘパリン/ mlのステップ2.1で説明したように、分離中に使用する独立したクレブス - ヘンゼライト緩衝液を準備します。 4℃（表1調製液を参照）クールソリューション。表1に記載したように、虚血のシミュレーションでは、虚血溶液を調製。 </li><li> Ca 2+のプロトコルの場合-依存収縮、要求された最終濃度にクレブス-ヘンゼライト緩衝液にの Ca 2+ を追加します。 注：直接、その使用前に、これらの溶液を調製し、炭酸カルシウムの沈殿を避けるために、カルボゲンでGASS。使用する前に、32℃に予熱するソリューションを提供します。 </li></ol>乳頭切除手順の間に使用されるガス発生チューブと解剖皿の調製<ol><li>ソフトガスを接続します専門家の作業とガス接続（あるいは100％の酸素、カルボゲン）にチューブ（外径2〜4ミリメートル）を歌います。 </li><li>解剖皿の内側継手ガス発生管の閉じたリングを作成します。ガス発生管に連続発泡が保証されている数回穿刺。このガス放出管は、複数回使用することができます。 </li><li>ピンがそれに貼り付けることができるように、底部（0.5センチメートル厚）のシリコンエラストマーと解剖皿を準備します。この皿を数回使用することができます。 </li></ol>マウスの左前方乳頭筋の4.絶縁注：乳頭筋の分離を開始する前に、ガスラインが閉塞の明確であることを確認してください。 <ol><li>乳頭筋の準備を開始する前に、プロトコルセクション1-3で述べたように、臓器浴の設定を準備します。実験の日にすべてのソリューションを準備します。 </li><li>頸椎脱臼のaccordiによって（約8〜12週齢）のマウスを生け贄に捧げます専門家の作業と施設のガイドラインにNG。 </li><li>前足を固定して仰臥位で動物を修正し、解剖ボードに足を後肢、鋭い骨はさみで肋骨を切断することにより、両側の胸部の横を開いて、横方向のカットとダイアフラムを切りました。心膜を削除します。今心臓と大動脈をアクセス可能です。 </li><li>心臓に近い血管体幹の鈍ピンセットで心臓を修正して、肺と周囲の組織からハサミですぐに心を分離。 </li><li>酸素（ステップ2.2）にガスを供給した冷却調製液で満たされたペトリ皿に拍動している心臓を転送します。ハートビートとピンセットで心臓頂点に触れる介して、そっと心の収縮を刺激することを許可します。 </li><li>すぐに心臓が完全にexsanguinousで、冷却された調製液（ステップ2.2）を充填し、O 2ガスを供給解剖皿に移します。使用上のこのステップ実体顕微鏡から。 </li><li>ょんを修正心が背側から見られるように右心室を通って小さなピンとRT（右心室は、オペレータのビューから右側に位置しています）。 </li><li>顕微はさみを使用すると、房室レベルで両方の心房を分離心室から組織を接続切断して捨てます。任意の機械的な圧力を避ける心尖にAV-バルブレベルから心室壁を通してカットを実行します。 </li><li>心室を開き、鉗子で左側の自由壁を固定し、このように両方の左心室乳頭筋を見てみました。 </li><li>少し乳頭筋の準備の弁膜帆の一部を保存左前乳頭筋の両側の心室組織の横を離れてカットし、触れないように注意してくださいまたは、できるだけ多くの乳頭筋をストレッチ。乳頭筋からの残りの心室壁組織を解剖。 </li><li>乳頭筋の準備の両側に取り付け絹糸（7/0、メートル法0.5）aration、弁膜帆上の1つの筋肉の部分に1つ。温度32°Cが提案されているように、臓器浴溶液を充填し、カルボゲンでガスを供給した器官槽室で準備を修正。 </li></ol>乳頭筋の5平衡と刺激<ol><li>直ちに臓器浴中で固定した後、1ヘルツの刺激周波数の矩形パルス（2ミリ秒の持続時間および100 mAの電流）と乳頭筋調製物を刺激します。 </li><li>使用オーガンバスタイプの設定に応じて連続的に変化したり、頻繁に手動で変更（5分ごと）のいずれかによって、臓器浴溶液の頻繁な変更を確認してください。 </li><li>徐々に達しにおける最大単収縮力まで、プレテンションを上げます。プリテンションの増加は単収縮力のさらなる増強が続いていないときに最大単収縮力に達します。 </li><li>平衡の45〜60分後、実験プロトコルを開始します。 </li></ol>注：以下のとおり実験プロトコルは、生理学的および病態生理学的条件の下で心臓の収縮を特徴づけるために、標準的な操縦を含みます。代表的なセクションでは、詳細には、これらのプロトコルを記述し、また（表2参照）代表的な結果を示します。 <ol><li>実験プロトコルの記録と分析<ol><li>記録のために、十分な時間分解能（取得率≥1kHzの）で、適切なソフトウェアを使用しています。 </li><li>単収縮力の大きさ（高さ）、ソフトウェアプログラムで述べたようにテンション（TTP）と最大値の半分緩和時間（それぞれ、R 50 / TFallを）ピークまでの時間（ 例として図4を参照してください 、ADInstrumentsの5.5チャート）などのパラメータを分析します。 </li></ol></li></ol>

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著者は、彼らが競合する金融利害関係を有していないことを宣言します。

本稿では、我々は、マウスの心臓生理学と病理学に関連するいくつかの科学的な質問に答えるだけでなく、トランスジェニック系統の分析と新たな医薬品のアプローチの発見をサポートするために使用することができ、in vitroでマウス乳頭筋の収縮性を調査する方法を説明心臓の機能不全を治療することができます。我々は、心臓の筋肉の収縮の、生理的な病理学的および薬理学的特?...

心臓の収縮のために自分の役割を参照するイオンチャネルのようなタンパク質の調査は異なる発症機序を発見し、そのような虚血および心不全などの心臓疾患のための新しい治療戦略を確立することが不可欠です。 哺乳動物の心筋細胞の収縮機能は、種々のイオンチャネル、輸送体および他のタンパク質によって調節することが知られています。活動電位は、Ca 2+チャネルは、細胞外空間からのCa 2+流入につながる電圧依存筋細胞膜のL型の活性化を誘発し、その後のCa 2+の細胞収縮2をトリガのCa 2+放出（CICR）1を 、誘発しました。生理学的または病理学的ストレスに対する心臓の収縮と適応に中心的な役割を果たしている-signaling の Ca 2+。カテコールアミンは、このようにキャンプを合成するアデニル酸シクラーゼ（AC）を刺激する、心臓βアドレナリン受容体を活性化します。活性化され、protein個のCa 2+トランジェントと心収縮の1,3,4の変更の結果、L型Ca 2+チャネル 、ホスホランバンとリアノジン受容体のような異なる細胞内および細胞膜結合タンパク質をリン酸化する（PKA）のキナーゼ。 cAMPはホスホジエステラーゼ（PDE）によって分解されます。 βアドレナリン受容体以外のGs共役受容体の活性化はまた、cAMPの蓄積をもたらします。 孤立した心室筋ストリップで収縮測定の技術が十分に大きい哺乳動物種5-8のために確立されています。マウスにおいて、遺伝子ターゲティングの可能性に基づいて、マウス心臓の生理機能を分析するための方法を確立することが重要です。しかし、マウスでは孤立した筋肉製剤の生理学的特性に関する既存のデータは、実験条件9-12によって異なります。 記載された方法は、左心室の乳頭筋の事前の心収縮性を分析するために使用されin vitroで parations。心収縮の調査は、血圧、神経液刺激および物理的または代謝的ストレスのようなin vivoでの心臓収縮を、修正の影響の非存在下で行われます。収縮筋調製物の叩解速度が厳密に定義され、任意に変更することができます。単収縮力は周波数または温度を破って、そのようなカルシウム濃度のような特定の刺激との関連で分析することができます。また、この方法は、異なるシグナル伝達経路の成分を調査すると、上記の実験条件を制御することにより、遺伝的に改変されたマウスモデルの心臓の性能を比較するために使用することができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P 5655</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F3917</td>
			<td>Hazard statement H312, solve in DMSO</td>
		</tr>
		<tr>
			<td>3-?Isobutyl-?1-?methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>Hazard statement H 302, solve in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>I5627</td>
			<td>Hazard statement H 315-H319-H335</td>
		</tr>
		<tr>
			<td>Sodium Heparine 250.000 IE/10ml</td>
			<td>ratiopharm</td>
			<td>PZN 3874685</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H7250</td>
			<td>Hazard statement H 315-H 317-H319- H334-H335</td>
		</tr>
	</tbody>
</table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

成体マウスの心臓から単離された乳頭筋は異なる生理学的/病理学的状態の間の心収縮性を研究するために使用することができます。収縮特性は、独立して、血管の緊張または神経液性状態などの外部の影響を評価することができます。これは、単離された心筋細胞を持つ単一セル測定の間及び心エコー検査などのインビボ研究で科学的なアプローチを示しています。従って、乳頭筋調製物は、心臓の生理機能/病態生理学を研究するための優れたモデルとして機能し、薬剤による調節またはトランスジェニック動物モデルの探査などの調査のために使用することができます。ここでは、器官槽の設定における心収縮性を調査するために、マウスの左前方の乳頭筋を単離する方法について説明します。心室壁から単離された筋肉片の調製とは対照的に、乳頭筋は、筋TISSUに損傷を与えることなく、 全体として調製することができます。E深刻。器官槽の設定は、複数の温度制御され、毒ガスと電極を装備した器官槽室で構成されています。孤立した乳頭筋は、器官槽室に固定され、電気的に刺激されます。誘発単収縮力は、圧力変換器と、このような力の大きさをけいれんおよび動態を分析する単収縮のようなパラメータを使用して記録されています。異なる実験プロトコルは、カルシウムおよび周波数依存収縮ならびにカテコールアミンや他の医薬品などの収縮剤の用量反応曲線を調査するために行うことができます。さらに、急性虚血のような病理学的状態をシミュレートすることができます。

単離されたマウス乳頭筋調製物の収縮の測定のために、この原稿のプロトコルは、生理学的条件下で再現性のある実験の結果を達成するために最適な条件に調整されます。最適な実験条件を定義するために、我々は、器官浴温度および細胞外カルシウム濃度（12も参照）に変化するパイロット実験を行いました。ここで説明するプロトコルは、1.5mMの細胞外カルシウム濃度及?...

Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

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

マウスの左心室の乳頭筋は 、インビトロで心筋収縮性を調べるために使用することができます。この記事では詳細に心臓収縮特性を研究するためのアイソレーションと実験プロトコルを説明しています。

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 ...

contractility-measurements-on-isolated-papillary-muscles-for

Research

JoVE Journal

Biology

14.8K ビュー.  Universität Heidelberg. マウスの左心室の乳頭筋は 、インビトロで心筋収縮性を調べるために使用することができます。この記事では詳細に心臓収縮特性を研究するためのアイソレーションと実験プロトコルを説明しています。 

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

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

免疫不全マウスにおける生体工学ヒト微小血管ネットワーク

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

生物学

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 ...

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, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

末梢血幹細胞移動の解析のためのマウス精巣挙筋で微小循環の生体顕微鏡

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

マウス単離された灌流腎臓テクニック

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

シンプルで効率的なマウスの心筋内注入による生体内の心臓特異的遺伝子操作法

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

電気生理学的研究のための成体、幼若、幼虫および胚性ゼブラフィッシュからの心臓および血管平滑筋細胞の単離(英語)

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Terminal H-reflex Measurements in Mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

マウスにおける終末H反射測定

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ,&#160;in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH.

単離灌流マウス心臓における過分極[1-13 C]ピルビン酸および13C/31P NMR分光法による心臓代謝の調査(英語)

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial ...

hiPSC-CMにおけるカルシウムおよび収縮性パラメータのリアルタイム測定

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

著者スポットライト:ヒト誘導多能性幹細胞由来心筋細胞におけるカルシウムおよび収縮性パラメータのリアルタイム測定  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

ヒト人工多能性幹細胞由来心筋細胞におけるカルシウムおよび収縮性パラメータのリアルタイム測定

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...

Methods Article

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

Methods Article

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