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>

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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><tbody><tr><td>Leica VT 1000s</td><td>Leica Microsystems, Wetzlar, Germany</td><td></td><td>Microtome with vibrating blade.</td></tr><tr><td>Stainless Steel Blades</td><td>Campden Instruments, Loughborough, England</td><td>7550-1-SS</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Sigma-Aldrich, St. Louise, USA</td><td>Z627992</td><td></td></tr><tr><td>Fine brush, &lt;em&gt;e.g.&lt;/em&gt; size 6 (4/32&quot;)</td><td>VWR, International, Radnor, USA</td><td>149-2125</td><td></td></tr><tr><td>Preparation table</td><td></td><td></td><td>self made</td></tr><tr><td>Molt for embedding ventricles in agarose</td><td></td><td></td><td>self made</td></tr><tr><td>1 mL Syringe</td><td>Becton, Dickinson; Franklin Lakes, USA</td><td>300013</td><td></td></tr><tr><td>27 G x 3/4`` Needles</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>20 G 11/2`` Needles</td><td>4657519</td><td></td><td></td></tr><tr><td>Small scissor</td><td>WPI, Sarasota, USA</td><td>501263</td><td></td></tr><tr><td>Tweezers #5, 0.1 x 0.06 mm tip</td><td>WPI, Sarasota, USA</td><td>500342</td><td></td></tr><tr><td>Oxygen gas (medical grade O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>Carbogen gas (95 % O&lt;sub&gt;2&lt;/sub&gt;, 5 % CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>7647-14-5</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746436</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746495</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>NIST200B</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, St. Louise, USA</td><td>51558</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S5761</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Sigma-Aldrich, St. Louise, USA</td><td>G8270</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>M7506</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S8045</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Henkel, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr><tr><td>Low-melt Agarose</td><td>Roth, Karlsruhe, Germany</td><td>6351.2</td><td></td></tr><tr><td>Heparin-sodium-25000 I.E./5 mL</td><td>Ratiopharm, Ulm, Germany</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX</td><td>ThermoScientific, Waltham, USA</td><td>10566016</td><td></td></tr><tr><td>SEC-10LX Amplifier</td><td>npi electronic GmbH, Tamm, Germany</td><td>SEC-10LX</td><td></td></tr><tr><td>EPC 9</td><td>HEKA Elektronik GmbH, Lambrecht, Germany</td><td></td><td></td></tr><tr><td>Zeiss Axiovert 200</td><td>Zeiss, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Low magnification Micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>Nm-3</td><td></td></tr><tr><td>High magnification, three-axis micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>MHW-3</td><td></td></tr><tr><td>Peristaltic perfusion pump</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>PPS2</td><td></td></tr><tr><td>2-channel temperature controller</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>TCO02</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus Europe GmbH, Planegg, Germany</td><td>Grass SD9</td><td></td></tr><tr><td>Glass capillaries</td><td>WPI, Sarasota, USA</td><td>1B150F-1</td><td></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

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

高周波高分解能心エコー検査：虚血 - 再灌流マウス心臓における心筋ストレイン、収縮性、および僧帽弁閉鎖不全の非侵襲的な反復測定に関する最初の証拠

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

医学

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

げっ歯類の内側嗅内皮質の背腹軸機構の調査のための矢状スライスの作成

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

神経科学

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

前根刺激のための斜め脊髄スライスの調製

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

ヒトの神経ノチカルスライスの二重電圧およびカルシウム光学マッピングによる前臨床心臓電気生理学的評価

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

生物工学

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

生理学的条件下での豚の心臓のスライスと培養

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

循環器疾患大型動物モデルにおける心臓の解剖技術と組織学的サンプリング

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

長期培養のためのヒト心筋組織の作製

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

ビブラートムカット心筋切れ切り切れからヒト心室心筋細胞を分離

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

生物学

Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue

Source: Ou, Q. et. al., <a href="https://www.jove.com/v/60913">Slicing and Culturing Pig Hearts under Physiological Conditions.</a> J. Vis. Exp. (2020)
This video demonstrates a vibratome-based procedure to generate viable, ultra-thin sections of pig heart. The prepared tissue slices can be cultured and used as model systems for pharmacological testing.

ブタ心室スライスの生成:ブタ心臓組織の超薄スライスを調製するためのビブラトームベースの技術

Source: Ou, Q. et. al., Slicing and Culturing Pig Hearts under Physiological Conditions. J. Vis. Exp. (2020)
This video demonstrates a vibratome-based ...