我们承认研讨会和神经生理学研究所的动物设施提供的支持。这项工作得到了Walter und Marga Boll-Stiftung，KölnFortune和Dezscher Stfung Herzforschung的支持。

动物处理必须符合当地动物福利委员会的指导方针和欧盟议会的2010/63 / EU指令。 准备解决方案<ol><li>制备没有Ca 2+ （组成为mM）的Tyrode溶液:NaCl 136，KCl 5.4，NaH 2 PO 4 0.33，MgCl 2 1，葡萄糖10，HEPES 5,2,3-丁二酮单肟（BDM）30。将pH调节至7.4用NaOH在4℃。 </li><li>用Ca 2+ （组成为mM）制备Tyrode溶液:NaCl 136，KCl 5.4，NaH 2 PO 4 0.33，MgCl 2 ，葡萄糖10，HEPES 5，BDM 30，CaCl 2 0.9。在4℃用NaOH调节pH至7.4。 </li><li>准备4％低熔点琼脂糖:将0.6 g低熔点琼脂糖置于15 mL Tyrode溶液中，不含Ca 2+ 。将混合物在微波炉中以750W加热10次，直到琼脂糖溶解。将溶液保持在37℃的恒定温度，＃176; C并连续搅拌。 </li><li>在37℃下保持不含血清的Dulbecco&#39;s改良的Eagle培养基（DMEM），用碳霉素（5％CO 2，95％O 2 ）鼓泡。 </li></ol> 2.准备切片机<ol><li>打开切片机。 </li><li>用冰将外切片机室填满。 </li><li>用冰冷的Tyrode溶液填充内切片机室，不含Ca 2+ ，并持续用100％O 2使溶液充氧。 </li><li>将钢刀片放入切片机刀片架上。 </li></ol> 3.小鼠心脏隔离<ol><li>皮下注射2500 U肝素。等待15分钟</li><li>通过颈椎脱位牺牲动物。 </li><li>通过胸骨切开术打开胸部。 </li><li>用小剪刀和镊子＃5仔细分析心包。 </li><li>将插管插入升主动脉，并用冰冷的Tyrode&#39;s 原位灌注冠状动脉不含Ca 2+的溶液，直到除去剩余的血液。 </li><li>用镊子和剪刀轻轻切除心脏，将冰块转移到冰冷的Tyrode溶液中，不含Ca 2+ 。 </li><li>用手术刀或剪刀将心房与心室分开。 </li></ol> 4.将心室嵌入4％低熔点琼脂糖中<ol><li>将顶点面朝上放置在琼脂糖模具中（ 图1 ）。将引脚放在模具中间的左心室。 </li><li>在37℃下用4％低熔点琼脂糖填充模具，直到心脏完全被覆盖。 </li><li>将模具放在冰上，以更快地硬化琼脂糖，防止组织漂浮。 </li><li>用手术刀从模具中取出含有心室的琼脂糖块。 </li><li>将块颠倒倒置并充满心室和琼脂糖块背面的间隙，由蜕皮的针留下，用4％的低熔点琼脂糖，使用20 G针的注射器。 </li><li>用手术刀修剪琼脂糖块，实现块的平坦底部和心脏顶点的直立位置。 </li></ol> 5.切片心脏组织<ol><li>用一滴氰基丙烯酸酯胶将块固定在切片机的样品架上。面向心脏顶点向上。 </li><li>将样品架放入切片机的内部样品室中，该样品室装满冰冷的Tyrode，不含Ca 2+ 。用Tyrode的解决方案完全覆盖琼脂糖块。 </li><li>根据进一步的应用（锋利的电极记录150-200μm）准备厚度为150-400μm的短轴切片，将刀片的振动频率保持在60-70 Hz，并将刀片向前移动尽可能慢。 </li><li>用细毛刷小心地从切片中取出剩余的琼脂糖。 </li><li>轻轻地输r片用巴斯德吸管浸入含有100％O 2的 0.9mM Ca 2+的Tyrode溶液中，并在冰上储存至少30分钟，以从切片程序中回收。 </li><li>然后，在37℃的DMEM中保持切片30分钟，用碳水充气，以便进一步使用之前洗去BDM。 </li></ol> 6.准备夏普电极设置<ol><li>在预热之前，在录制开始前30分钟打开所有电器。 </li><li>将3厘米的细胞培养皿放在放置在倒置显微镜上的加热板上。 </li><li>将定制的环形电极（ 图2 ）放入盘中，并连接前置放大器和刺激电极的接地线。 </li><li>将灌注系统的柔性管连接到盘上。 </li><li>用DMEM填充灌注系统的储存器，用碳水充气。 </li><li>打开灌注泵并设置灌注量te至2-3 mL / min。 </li><li>通过调节流量加热器和加热板将盘中DMEM的温度调节至37°C。 </li></ol> 7.行动潜在记录<ol><li>将心室切片放入装有DMEM的盘中。 </li><li>用倒置显微镜检查组织的结构完整性和活力（基于收缩功能）。 </li><li>用3 M KCL填充记录玻璃电极。 </li><li>用DMEM填充刺激电极。 </li><li>将记录电极和刺激电极放在电极支架上。 </li><li>将刺激电极仔细放在切片上并打开电刺激器。以1-2赫兹的刺激频率开始。 </li><li>将记录电极用显微操纵器移动到预期的记录位置。 </li><li>缓慢降低记录电极直到尖端接触组织。 </li><li>应用短矩形电脉冲记录电极穿透细胞膜。 </li><li>仔细重新定位记录电极，直到确保稳定的信号。 </li><li>开始记录动作电位。 </li></ol>

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</ol>

作者没有任何声明。

心室切片使电生理，药理和机械研究具有体内保留的组织结构，并可将测量技术直接进入心脏的所有区域。已经在胚胎，新生儿和成人切片9,10,11中证明了生理作用潜力。生物活性染色证实切片的活力除了切片程序直接损伤的表层外，其余的活性染色9 。使用尖锐玻璃电极的切片内的动作电位记录可用于研究如本文所述的移植心肌细胞的整合和成熟，或加入心脏活性?...

由于Yamamot和Mcllwain于1966年在体外证实了脑片的电活动，所以在基础科学中经常使用薄片切片1 。此后，从脑2 ，肝3 ，肺4和心肌组织5,6,7的切片进行了电生理和药理学研究。 1990年8月描述了来自新生大鼠心脏的心室切片中的第一次膜片钳记录，但是这种技术被遗忘了一段时间。十多年后，我们组织制定了一种新的方法来准备鼠胚胎9 ，新生儿10和成人11心脏切片。这些活组织切片可用于急性实验（成人切片）s可以培养数小时）或短期培养实验（胚胎和新生儿切片可以培养几天）。切片在体内显示如电生理特征和均匀的激发扩散，如通过尖锐电极动作电位和微电极阵列记录所评估的。由于其"二维"形态，它们允许记录电极直接进入心室的所有区域，这使得它们成为电生理调查的有趣工具，并且与Langendorff灌注的整个心脏相比提出了新的实验选择。离子通道阻滞剂如维拉帕米（L型Ca 2+通道阻断剂），利多卡因（Na +通道阻断剂），4-氨基吡啶（非选择性电压依赖性K +通道阻滞剂）和亚麻酸（KCNQ K +通道阻塞） 9，11 </sup&gt;对应于已解离的心肌细胞的已知作用。等轴力测量显示正的力频率关系，强烈建议完整的收缩功能10 。这些研究结果表明，鼠心室切片适合作为生理和药理学研究的体外组织模型。此外，受体心脏的心室切片与尖锐的电极记录结合已经被证明是一个非常有用的工具，用于表征移植胎儿12,13,14和干细胞衍生的15个心肌细胞的电和机械整合以及成熟。 总之，心室切片是一种有价值且成熟的多细胞组织模型，应被认为与分离的心肌细胞和Langendorff灌注心脏互补在心血管研究中，提供体内类似组织结构（与分离的细胞相反）的主要优点，以及直接访问诸如尖锐电极记录到心脏的所有区域的测量技术（与全心脏制剂相反）。

<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

鼠心肌细胞已广泛用于心脏生理学和新治疗策略的体外研究。然而，解离的心肌细胞的多细胞制剂并不代表心肌细胞，非肌细胞和细胞外基质的复合体内结构，其影响心脏的机械和电生理学性质。在这里，我们描述了一种技术，用于制备具有保留的体内类似组织结构的成年小鼠心脏的活动性心室切片，并且证明其适用于电生理记录。切除心脏后，将心室与心房分离，用含有2,3-丁二酮一肟的无Ca 2+溶液灌注，并包埋在4％ 低熔点琼脂糖块中。将块放置在具有振动叶片的切片机上，并且制备厚度为150-400μm的组织切片，保持振动频率60-70 Hz的刀片速度，并尽可能缓慢地向前移动刀片。切片的厚度取决于进一步的应用。切片储存在冰冷的Tyrode溶液中，用0.9mM Ca 2+和2,3-丁二酮一肟（BDM）处理30分钟。之后，将切片转移到37℃的DMEM中30分钟以洗出BDM。切片可用于具有尖锐电极或微电极阵列的电生理研究，用于力测量分析收缩功能或调查移植的干细胞衍生的心肌细胞和宿主组织的相互作用。对于尖锐的电极记录，将切片置于倒置显微镜的加热板上的3cm细胞培养皿中。用单极电极刺激切片，用锋利的玻璃电极记录切片内心肌细胞的细胞内动作电位。

心肌梗死导致心肌细胞几乎不可逆的损失。使用干细胞衍生的心肌细胞进行外源性心脏再生的细胞替代疗法是一种有希望的治疗方法。移植细胞的电气整合和成熟对细胞替代疗法的安全性和效率至关重要。 为了评估整合和成熟，我们将来自表达增强的绿色荧光蛋白（eGFP）的诱导多能干细胞（iPSCM; 2次注射0.5×10 6 iPSCM /1...

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

在这里，我们描述了成年小鼠可行心室切片的制备及其对尖锐电极动作电位记录的应用。这些多细胞制剂提供了保留在体内的类似组织结构，这使得它们成为体外电生理和药理学研究的有价值的模型。

鼠短轴心脏心脏切片用于电生理研究

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 次观看.  University of Cologne. 在这里，我们描述了成年小鼠可行心室切片的制备及其对尖锐电极动作电位记录的应用。这些多细胞制剂提供了保留在体内的类似组织结构，这使得它们成为体外电生理和药理学研究的有价值的模型。 

视频: 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.

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

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

科研

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.

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

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

神经科学

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.

斜脊髓切片用于神经前根刺激的制备

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.

成年小鼠视网膜电生理研究水平切片的制备

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.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

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

生物工程

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.

生理条件下猪心的切片和培育

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.

心血管疾病大型动物模型中心脏的解剖技术和组织学取样

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.

长期培养用人心肌组织的制备

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.

人体心室心肌细胞与颤音-切切心肌切片分离

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.

猪脑室切片的生成:一种基于振动切片机的技术，用于制备猪心脏组织的超薄切片

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

鼠短轴心脏心脏切片用于电生理研究

摘要

探索更多视频

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备

斜脊髓切片用于神经前根刺激的制备

成年小鼠视网膜电生理研究水平切片的制备

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

生理条件下猪心的切片和培育

心血管疾病大型动物模型中心脏的解剖技术和组织学取样

长期培养用人心肌组织的制备

人体心室心肌细胞与颤音-切切心肌切片分离

猪脑室切片的生成:一种基于振动切片机的技术，用于制备猪心脏组织的超薄切片

鼠短轴心脏心脏切片用于电生理研究

摘要

探索更多视频

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

斜脊髓切片用于神经前根刺激的制备

成年小鼠视网膜电生理研究水平切片的制备

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

生理条件下猪心的切片和培育

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