我々は、神経生理学研究所のワークショップと動物施設が提供する支援を認めています。この作品は、Walter und Marga Boll-Stiftung、KölnFortune、Deutsche StiftungfürHerzforschungによって支持されました。

動物の取扱いは、地域の動物福祉委員会の指針と、欧州議会の2010/63 / EU指令に適合していなければならない。 1.ソリューションの準備<ol><li> NaCl 136、KCl 5.4、NaH 2 PO 4 0.33、MgCl 2 1、グルコース10、HEPES 5,2,3-ブタンジオンモノオキシム（BDM）30を用いて、Ca 2+ （mMの組成）を含まないTyrode溶液を調製する。 4℃でNaOHで処理した。 </li><li> NaCl 136、KCl 5.4、NaH 2 PO 4 0.33、MgCl 2 1、グルコース10、HEPES 5、BDM 30、CaCl 2 0.9のCa 2+ （mM単位の組成）でTyrodeの溶液を調製する。 4℃でNaOHでpHを7.4に調整する。 </li><li> 4％低融点アガロースの調製：Ca 2+を含まないTyrode溶液15 mLに低融点アガロース0.6 gを入れます。アガロースが溶解するまで、混合物を電子レンジで750Wで10-15秒間2回加熱する。溶液を37℃の一定温度に保ち、＃176; Cを添加し、連続的に攪拌する。 </li><li> 37℃で血清を含まないダルベッコ改変イーグル培地（DMEM）をカルボゲン（5％CO 2、95％O 2 ）で泡立てた状態に保つ。 </li></ol> 2.ミクロトームの準備<ol><li>ミクロトームのスイッチを入れる。 </li><li>外側のミクロトームチャンバーを氷で満たします。 </li><li>内部ミクロトームチャンバーをCa 2+を含まない氷冷Tyrode溶液で満たし、溶液を100％O 2で連続的に酸素化する。 </li><li>スチールブレードをミクロトームのブレードホルダーに置きます。 </li></ol> 3.マウス心臓の分離<ol><li> 2,500Uのヘパリンを皮下に注射する。 15分待ちます。 </li><li>頚椎脱臼によって動物を犠牲にする。 </li><li>胸骨を開ける。 </li><li>慎重に小さなはさみと鉗子＃5を使用して心膜を切開する。 </li><li>上行大動脈にカニューレを挿入し、 その場で冠状動脈を氷で冷やすTyrode&#39;s残りの血液が取り除かれるまで、Ca 2+を含まない溶液である。 </li><li>穏やかに鉗子とはさみで心臓を切除し、冷たいタイロードの溶液をCa 2+なしで乗せて移します。 </li><li>メスまたはハサミで心房を心室から分離する。 </li></ol> 4. 4％低融点アガロースへの心室の埋め込み<ol><li>アペックスを上向きにして心室をアガロースモールドに置きます（ 図1 ）。左心室の金型の中央にピンを置きます。 </li><li>心臓が完全に覆われるまで、37℃で4％低融点アガロースでモールドを満たす。 </li><li>金型を氷上に置き、アガロースのより速い硬化が組織の浮きを防止するようにする。 </li><li>メスを用いて、心室を含むアガロースブロックを金型から取り出す。 </li><li>ブロックを裏返しにして、心室と、アガロースブロックの裏側の隙間を満たします。20Gの針を備えたシリンジを用いて4％低融点アガロースを用いてモルトのピンによって放置された。 </li><li>メスでアガロースブロックをトリムして、ブロックの平坦な底部および心尖部の直立位置を達成する。 </li></ol> 5.心室のスライス<ol><li>シアノアクリレート接着剤を滴下してミクロトームの試料ホルダーにブロックを固定する。心尖部を上に向けます。 </li><li>標本ホルダーをミクロトームの内部標本チャンバーに置き、Ca 2+を含まない氷冷タイロードで満たします。タイロードの溶液でアガロースブロックを完全に覆います。 </li><li>さらなる適用（シャープな電極記録150-200μmの場合）に応じて150〜400μmの厚さの短軸スライスを準備し、ブレードの振動周波数を60〜70Hzに保ち、できるだけゆっくりとブレードを前進させる。 </li><li>細かいブラシを使用して、残りのアガロースをスライスから慎重に除去します。 </li><li>優しくトランスファー0.9mMのCa 2+を含むTyrode溶液にパスツールピペットでスライスし、100％O 2を通気し、スライス手順から回復するために氷上で少なくとも30分間貯蔵する。 </li><li>その後、37℃でDMEM中で30分間スライスし、カルボジェンを通気し、その後の使用の前にBDMを洗い流す。 </li></ol> 6.シャープな電極セットアップの準備<ol><li>予熱のために、録音が始まる30分前にすべての電気装置をオンにします。 </li><li> 3cmの細胞培養皿を、倒立顕微鏡上に置かれた加熱プレート上に置く。 </li><li>ディッシュにカスタムメイドのリング電極（ 図2 ）を置き、プリアンプと刺激電極のアース線を接続します。 </li><li>灌流システムのフレキシブルチューブを皿に接続します。 </li><li> carbogenを通気したDMEMで灌流システムのリザーバを満たしてください。 </li><li>灌流ポンプのスイッチを入れ、潅流を設定する2~3mL /分である。 </li><li>フローヒーターと加熱プレートを調節して、ディッシュ内のDMEMの温度を37°Cに調整します。 </li></ol> 7.アクション潜在的なレコーディング<ol><li> DMEMで満たされた皿に心室スライスを置く。 </li><li>倒立顕微鏡を用いて組織の構造的完全性および生存能力（収縮機能に基づく）をチェックする。 </li><li>記録用ガラス電極に3M KCLを充填する。 </li><li>刺激電極をDMEMで満たす。 </li><li>記録電極と刺激電極を電極ホルダーの上に置く。 </li><li>スライス上に刺激電極を注意深く置き、電気刺激装置をオンにします。 1〜2Hzの刺激周波数で開始する。 </li><li>マイクロマニピュレータを使用して記録電極を目的の記録位置に移動します。 </li><li>先端が組織に触れるまで、記録電極をゆっくりと下げる。 </li><li>短い長方形の電気パルスを記録電極が細胞膜を貫通する。 </li><li>安定した信号が得られるまで記録電極の位置を慎重に変えてください。 </li><li>活動電位の記録を開始する。 </li></ol>

<ol>
	<li>Yamamoto, C., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activities+in+thin+sections+from+the+mammalian+brain+maintained+in+chemically-defined+media+in+vitro.">Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro.</a> J Neurochem. 13, 1333-1343 (1966).</li><li>Colbert, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cortical+brain+slices+for+electrophysiological+recording.">Preparation of cortical brain slices for electrophysiological recording.</a> Methods Mol Biol. 337, 117-125 (2006).</li><li>Ad Graaf, I., Groothuis, G. M., Olinga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+tissue+slices+as+a+tool+to+predict+metabolism+of+novel+drugs.">Precision-cut tissue slices as a tool to predict metabolism of novel drugs.</a> Expert Opin Drug Metab Toxicol. 3, 879-898 (2007).</li><li>Kim, Y. 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=Cardiopulmonary+toxicity+of+peat+wildfire+particulate+matter+and+the+predictive+utility+of+precision+cut+lung+slices.">Cardiopulmonary toxicity of peat wildfire particulate matter and the predictive utility of precision cut lung slices.</a> Part Fibre Toxicol. 11, 29 (2014).</li><li>Nembo, E. N., 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=In+vitro+chronotropic+effects+of+Erythrina+senegalensis+DC+(Fabaceae)+aqueous+extract+on+mouse+heart+slice+and+pluripotent+stem+cell-derived+cardiomyocytes.">In vitro chronotropic effects of Erythrina senegalensis DC (Fabaceae) aqueous extract on mouse heart slice and pluripotent stem cell-derived cardiomyocytes.</a> J Ethnopharmacol. 165, 163-172 (2015).</li><li>Wang, 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=Cardiac+tissue+slices:+preparation,+handling,+and+successful+optical+mapping.">Cardiac tissue slices: preparation, handling, and successful optical mapping.</a> Am J Physiol Heart Circ Physiol. 308, H1112-H1125 (2015).</li><li>Bussek, A., 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=Tissue+slices+from+adult+mammalian+hearts+as+a+model+for+pharmacological+drug+testing.">Tissue slices from adult mammalian hearts as a model for pharmacological drug testing.</a> Cell Physiol Biochem. 24, 527-536 (2009).</li><li>Burnashev, N. A., Edwards, F. A., Verkhratsky, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recordings+on+rat+cardiac+muscle+slices.">Patch-clamp recordings on rat cardiac muscle slices.</a> Pflugers Arch. 417, 123-125 (1990).</li><li>Pillekamp, F., 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=Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation.">Establishment and characterization of a mouse embryonic heart slice preparation.</a> Cell Physiol Biochem. 16, 127-132 (2005).</li><li>Pillekamp, F., 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=Neonatal+murine+heart+slices.+A+robust+model+to+study+ventricular+isometric+contractions.">Neonatal murine heart slices. 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著者は宣言することは何もない。

心室スライスは、組織構造のような保存されたインビボでの電気生理学的、薬理学的および機械的研究ならびに心臓のすべての領域への測定技術の直接アクセスを可能にする。生理学的作用潜在的特性は、胚、新生児および成人のスライス9,10,11において示されている。スライシング手順によって直接損傷された表面層を除いて、スライスの活性は、活力染色によって確認された<sup clas...

YamamotとMcllwainは1966年に脳切片の電気的活動がin vitroで維持されていることを示して以来、薄い組織切片が基礎科学で頻繁に使用されています1 。それ以来、脳2 、肝臓3 、肺4および心筋組織5,6,7の切片について電気生理学および薬理学的研究が行われてきた。新生児ラット心臓からの心室スライスにおける最初のパッチクランプ記録は1990年8月に記載されていたが、この技術はしばらくの間忘れ去られた。 10年以上後、我々のグループは、マウス胚9 、新生児10および大人11の心臓スライスを調製するための新しい方法を確立した。これらの生存可能な組織切片は、急性実験（成人スライス数時間栽培することもできる）または短期間の培養実験（胚および新生児スライスを数日間培養することができる）。スライスは、電気生理学的特徴および鋭い電極活動電位およびマイクロ電極アレイ記録11によって評価される均一な励起広がりのようなインビボで示す 。 「2次元」形態のため、脳室内のすべての領域に記録電極を直接アクセスすることができ、電気生理学的調査のための興味深いツールとなり、Langendorff灌流心臓全体と比較して新しい実験的選択肢をもたらす。ベラパミル（L型Ca 2+チャンネルブロッカー）、リドカイン（Na +チャンネルブロッカー）、4-アミノピリジン（非選択的電圧依存性K +チャンネルブロッカー）およびリノパルジン（KCNQ K）のようなイオンチャネルブロッカーに対するスライスの薬物応答は、 +チャンネルブロッカー） 9,11 </sup&gt;は、解離した心筋細胞に対する既知の効果に対応した。等尺性力測定は正の力周波数関係を示し、無傷の収縮機能を強く示唆した10 。これらの発見は、マウス心室スライスが、生理学的および薬理学的研究のためのインビトロ組織モデルとして適切であることを実証した。さらに、鋭い電極記録と組み合わせたレシピエント心臓の心室スライスは、移植された胎児12,13,14および幹細胞由来の15の心筋細胞の電気的および機械的な統合ならびに成熟を特徴付ける非常に有用なツールであることが判明している。 要約すると、心室スライスは、価値ある、十分に確立した多細胞組織モデルであり、解離した心筋細胞およびランゲンドルフ灌流心臓に対して相補的であると考えられるべきである（解離した細胞とは対照的に） インビボのような組織構造を提供すること、および（心臓全体の準備とは対照的に）鋭い電極記録のような測定技術の直接的なアクセスを心臓の全ての領域に提供することの大きな利点がある。

<table border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Leica VT 1000s</td>
			<td style="width:340px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;">Microtome with vibrating blade.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Stainless Steel Blades</td>
			<td style="width:340px;">Campden Instruments, Loughborough, England</td>
			<td>7550-1-SS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes&#160;</td>
			<td>Sigma-Aldrich, St. Louise, USA</td>
			<td>Z627992&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Fine brush, e.g. size 6 (4/32&#34;)</td>
			<td style="width:340px;">VWR, International, Radnor, USA</td>
			<td>149-2125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Preparation table</td>
			<td style="width:340px;"></td>
			<td style="width:140px;"></td>
			<td style="width:167px;">self made</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Molt for embedding ventricles in agarose</td>
			<td style="width:340px;"></td>
			<td style="width:140px;"></td>
			<td style="width:167px;">self made</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:248px;">1 ml Syringe</td>
			<td style="width:340px;">Becton, Dickinson;&#160; Franklin Lakes, USA</td>
			<td>300013</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">27Gx3/4`` Needles</td>
			<td rowspan="2" style="width:340px;">Braun, Melsungen, Germany</td>
			<td>4657705</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">20G 11/2`` Needles</td>
			<td>4657519</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Small scissor</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td>501263</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Tweezers #5, 0.1 x 0.06 mm tip</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td>500342</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen gas (medical grade O2)</td>
			<td style="width:340px;">Linde, Munich, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Carbogen gas (95 % O2, 5 % CO2)&#160;</td>
			<td style="width:340px;">Linde, Munich, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NaCL</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">7647-14-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">KCL</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">746436</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">CaCl2</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">746495</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">KH2PO4</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">NIST200B&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">HEPES</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">51558</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">NaHCO3</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">S5761&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">D(+)-Glucose</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">G8270&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">MgSO4</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">M7506&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NaOH</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">S8045&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Cyanoacrylate glue&#160;</td>
			<td style="width:340px;">Henkel, D&#252;sseldorf, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Low-melt Agarose&#160;</td>
			<td style="width:340px;">Roth, Karlsruhe, Germany</td>
			<td style="width:140px;">6351.2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Heparin-sodium-25000 I.E./5mL</td>
			<td style="width:340px;">Ratiopharm, Ulm, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM), high glucose, GlutaMAX</td>
			<td style="width:340px;">ThermoScientific, Waltham, USA</td>
			<td style="width:140px;">10566016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">SEC-10LX Amplifier</td>
			<td style="width:340px;">npi electronic GmbH, Tamm, Germany</td>
			<td>SEC-10LX</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">EPC 9</td>
			<td style="width:340px;">HEKA Elektronik GmbH, Lambrecht, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Zeiss Axiovert 200</td>
			<td style="width:340px;">Zeiss, Oberkochen, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">&#160;Low magnification Micromanipulator</td>
			<td style="width:340px;">Narashige, Tokyo, Japan</td>
			<td style="width:140px;">Nm-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">High magnification, three-axis micromanipulator</td>
			<td style="width:340px;">Narashige, Tokyo, Japan</td>
			<td style="width:140px;">MHW-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Peristaltic perfusion pump</td>
			<td style="width:340px;">Multi Channel Systems, Reutlingen, Germany</td>
			<td style="width:140px;">PPS2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">2-channel temperature controller</td>
			<td style="width:340px;">Multi Channel Systems, Reutlingen, Germany</td>
			<td style="width:140px;">TCO02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Square pulse stimulator</td>
			<td style="width:340px;">Natus Europe GmbH, Planegg, Germany</td>
			<td style="width:140px;">Grass SD9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Glass capillaries</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td style="width:140px;">1B150F-1</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

murine short axis ventricular heart slices for electrophysiological studies

マウス心筋細胞は、心臓生理学および新しい治療戦略のインビトロ研究に広く使用されている。しかし、解離した心筋細胞の多細胞調製物は、心臓の機械的および電気生理学的特性の両方に影響を与える、心筋細胞、非心筋細胞および細胞外マトリックスの生体内構造の複合体を代表するものではない。ここでは、保存されたインビボのような組織構造を有する成体マウス心臓の実行可能な心室スライスを調製する技術を説明し、電気生理学的記録のためのそれらの適合性を実証する。心臓の切除後、脳室を心房から分離し、2,3-ブタンジオンモノオキシムを含有するCa 2+不含溶液で灌流し、4％ 低融点アガロースブロックに包埋する。このブロックを振動刃付きミクロトーム上に置き、厚さ150〜400μmの組織切片を作製し、振動をfreブレードを60-70Hzで回転させ、ブレードを可能な限りゆっくりと前進させる。スライスの厚さは、さらなる用途に依存する。スライスを、0.9mMのCa 2+および2,3-ブタンジオンモノオキシム（BDM）を含む氷冷Tyrode溶液に30分間保存する。その後、スライスを37℃のDMEMに30分間移してBDMを洗い流す。スライスは、収縮機能を分析するための力測定、または移植された幹細胞由来の心筋細胞と宿主組織との相互作用を調べるために、鋭利な電極または微小電極アレイを用いた電気生理学的研究に使用することができる。鋭い電極記録のために、スライスを倒立顕微鏡の加熱プレート上の3cm細胞培養皿に入れる。スライスを単極電極で刺激し、スライス内の心筋細胞の細胞内作用電位を鋭利なガラス電極で記録する。

心筋梗塞は、実質的に不可逆的な心筋細胞の喪失をもたらす。外因性心臓再生のための幹細胞由来心筋細胞を用いる細胞補充療法は有望な治療アプローチである。移植された細胞の電気的統合および成熟は、細胞置換療法の安全性および効率のために重要である。 組込みおよび成熟を評価するために、本発明者らは、成?...

Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

ここでは、成体マウスからの生存心室スライスの調製および鋭い電極活動電位記録のためのそれらの使用を記載する。これらの多細胞調製物は、 インビボでの電気生理学的および薬理学的研究のための貴重なモデルとなる組織構造のような保存されたインビボを提供する。

電気生理学的研究のためのマウス短軸心室スライス

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

murine-short-axis-ventricular-heart-slices-for-electrophysiological

Research

JoVE Journal

Medicine

7.3K ビュー.  University of Cologne. ここでは、成体マウスからの生存心室スライスの調製および鋭い電極活動電位記録のためのそれらの使用を記載する。これらの多細胞調製物は、 インビボでの電気生理学的および薬理学的研究のための貴重なモデルとなる組織構造のような保存されたインビボを提供する。 

ビデオ: Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Algisyl-LVRを使っハーツ失敗関数内で左心室壁応力と改善の削減

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

創傷治癒のマウスモデル

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心筋内細胞送達：マウスハーツの観測

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

ネズミ異心臓移植技術

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Use of Two Intracorporeal Ventricular Assist Devices As a Total Artificial Heart

Mechanical circulatory support (MCS) has been introduced as a viable alternative to heart transplantation primarily through the use of intracorporeal ventricular assist devices (VADs) for support of the left ventricle. However, certain clinical scenarios warrant biventricular mechanical support. One strategy for some patients is the excision of both ventricles and the implantation of two VAD pumps as a total artificial heart (TAH). This has recently been made possible by the improvements in device design and the small profile of centrifugal devices. This TAH approach remains experimental with many important challenges such as the device settings to balance the right and left circulation, the orientation of the devices and the outflow graft with their influence on hemolysis and stability, and the outcome of chronic support using such an orientation. This protocol aims to provide a reproducible approach for total artificial heart replacement with two intracorporeal centrifugal VADs in a cow model.

Here, we present a protocol using two centrifugal pumps as a total artificial heart replacement.

完全人工心臓として 2 つの体内心室補助装置の使用

Mechanical circulatory support (MCS) has been introduced as a viable alternative to heart transplantation primarily through the use of intracorporeal ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高解像度光学マッピングとマウスの心房の電気生理学的評価

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

元ヴィヴォ・ランゲンドルフ・パーフュード・ハーツの眼電図と電気生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

肝臓生物学のEx Vivoモデルとしてのマウス精密カット肝臓スライス

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

ネズミ星状神経節の位置、解剖、分析

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

Use of a Percutaneous Ventricular Assist Device/Left Atrium to Femoral Artery Bypass System for Cardiogenic Shock

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the left ventricle by draining blood from the left atrium (LA) and returning it to the systemic arterial circulation via the femoral artery. It can provide flows ranging from 2.5-5 L/min depending on the size of the cannula. Here, we discuss the mechanism of action of LAFAB, available clinical data, indications for its use in cardiogenic shock, steps of implantation, post-procedural care, and complications associated with the use of this device and their management.
We also provide a brief video of the procedural component of device therapy, including the pre-placement preparation, percutaneous placement of the device via transseptal puncture under echocardiographic guidance and the post-operative management of device parameters.

The following article describes the stepwise procedure for placement of a device (e.g., Tandemheart) in cardiogenic shock (CS) that is a percutaneous left ventricular assist device (pLVAD) and a left atrial to femoral artery bypass (LAFAB) system that bypasses and supports the left ventricle (LV) in CS.

心原性ショックに対する経皮心室補助装置/左心房から大腿動脈バイパスシステムへの使用

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the ...