作者感谢 Hartwig Wieboldt 出色的技术援助, 以及汉堡-离心大学医学中心的 UKE 显微成像设施 (Umif) 提供显微镜和支持。这项研究是由 Förderverein des Universitären Herzzentrums 汉堡 e 和 DZHK (德国心血管研究中心) [FKZ 81Z4710141] 资助的。

所有涉及动物的程序均由汉堡州、汉堡大学动物保育和使用委员会的地方当局批准。
1. Langendorff 装置的研制
注: 可使用商用 Langendorff 灌注系统。
<ol><li>制备改性克雷布斯-Henseleit 溶液 (119 毫米氯化钠、25毫米碳酸氢钠、4.6 毫米氯化钾、1.2 毫米磷酸钾磷酸钙、1.1 毫米硫酸镁、2.5 毫米氯化钙、8.3 毫米葡萄糖和2毫米钠)丙酮酸)。将 95% oxygen/5%carbon 的混合物添加到灌注液中, 以防止钙沉淀。用0.22 µm 的孔隙大小过滤缓冲区。</li>
	<li>添加一个药理剂到缓冲, 以调查其对心脏电生理和心脏交感神经的影响, 根据需要。</li>
	<li>启动水浴, 并将灌注液包括 95% oxygen/5%carbon 的混合物。调节水浴的温度, 使灌注液温度直接在套管前37˚C。</li>
	<li>当达到正确的温度后, 启动辊泵, 并在设备上装入灌注液。</li>
	<li>在连接心脏之前, 调整泵的速度, 这样在安装到设备上时, 套管内没有气泡被留下。</li>
</ol>2. 硬软件准备
<ol><li>将数字数据采集系统及其相应的软件连接到 Langendorff 设备, 用于连续记录灌注压力、流速和心率。<ol>
<li>将常规设置中的目标灌注压力设置为 80 mmHg。</li>
		<li>开始录制。</li>
	</ol>
</li>
	<li>使用电生理学导管 (材料表) 与铂电极, 电极表面 0.5 x 0.5 毫米, 和电极间距0.5 毫米的数据记录和刺激与指定的数字刺激发电机。<ol>
<li>将导管放在靠近心脏的部位, 将其放置到设备的附件中。</li>
		<li>通过从导管中选择2电极来准备刺激, 并使用100毫秒的周期长度。</li>
	</ol>
</li>
</ol>3. 心脏的准备
<ol><li>加入冷 (~ 2-4 ˚C) 灌注缓冲 (10-20 毫升和 40-50 毫升, 因此整个盘子被盖) 对2培养皿 (直径 6 cm 和 10 cm) 和盘与套管并且安置他们在冰直接地其次和在显微镜下。在套管周围准备一个双结。</li>
	<li>通过使用梅奥剪刀和窄模式钳打开胸腔, 快速切除颈椎脱位后的心脏。然后用伦敦钳夹住隔膜上方的主动脉和上腔静脉, 用斜视剪刀切开与脊柱相近的所有血管和结缔组织。</li>
	<li>将心肺块转移到第一道菜 (6 厘米直径), 用冰冷的缓冲器填满, 小心地取出肺部, 而不损害心脏, 使用斜视剪刀和伦敦钳。然后将心脏放在显微镜下, 用弹簧剪刀和 SS 钳仔细去除胸腺、食道和气管。</li>
	<li>用弹簧剪刀在右心房上部切开 1.5-2 毫米的孔, 插入导管。在肺动脉上切开一个洞。然后直接在 supraaortic 分支下切开主动脉, 从主动脉中取出组织, 使结能容易附着。</li>
	<li>保持心房脂肪垫, 包括主要 atrially 位于 ganglionated 树脂, 周围的左心房完整或完全删除他们仔细解剖。</li>
	<li>用套管把心脏转到盘子里, 把它放在显微镜下。将主动脉通过套管拉上, 并将准备好的结栓在主动脉周围。确保导管在主动脉中不放置太深, 使主动脉瓣膜和冠状动脉血管游离。</li>
	<li>将套管快速连接到 Langendorff 装置。请确保套管内没有气泡。</li>
	<li>将灌注压力转换为 80 mmHg, 允许恒定的压力灌注。</li>
	<li>将导管小心插入右心房和右心室, 不接触或损害心脏, 用胶带将导管连接到套管。</li>
	<li>开始刺激与准备的周期长度100毫秒的初始20分钟平衡期。</li>
	<li>关闭房间, 使温度稳定。</li>
</ol>4. 电生理参数和心脏交感神经
<ol><li>通过导管的远端或近端电极对心房或心室起搏阈值进行程序化刺激, 以评估电生理参数, 如以下步骤所述。<ol>
<li>确定窦节点恢复时间为十年代后 S1S1 周期长度为120毫秒、100毫秒和80毫秒的固定速率起搏后的最大返回周期长度。</li>
		<li>确定文氏点为最长的 S1S1 周期长度 (8 刺激;S1S1: 100 毫秒;2毫秒逐步减少), 损失 11 AV 节点传导。确定房室结节难治期为最长 S1S2 (12 项刺激;S1S1: 120 毫秒, 110 毫秒, 100ms;一个短耦合 extrastimulus 与2毫秒逐步 S1S2 减少) 与丧失 AV 节传导。</li>
		<li>确定心房和心室难治期为最长 S1S2 (12 刺激;S1S1: 120 毫秒, 110 毫秒, 100ms;一个短耦合 extrastimulus 与2毫秒逐步 S1S2 减少) 与缺席心房或心室反应10,11。</li>
	</ol>
</li>
	<li>执行编程 extrastimulation (S1S1: 120 毫秒, 100 毫秒, 80 毫秒, 其次是3额外节拍; 60-20 ms 以2毫秒逐步减少) 或爆裂起搏协议 (5 s 在 S1S1:50 毫秒以 10 ms 逐步减少) 与兰贝斯惯例符合 evaluate 心室心脏交感神经 10, 11, 12.</li>
</ol>5. 心外膜传导测量
注: 记录单极心外膜电图使用128通道, 计算机辅助记录系统, 采样率为25赫的高分辨率映射。使用32多电极阵列 (多边环境协定; 电极间距离: 300 µm; 1.8 x 1.8 毫米)。请注意, 数据是带通滤波的 (50 Hz) 和数字化的12位和信号范围 20 mV。
<ol><li>将多边环境协定置于心脏的指定区域, 并将接地添加到心脏的另一部分13,14,15。</li>
	<li>将心外膜刺激导管靠近多边环境协定, 并开始持续刺激。</li>
	<li>通过检查信号质量和振幅, 确认电极良好接触后开始记录。</li>
	<li>利用离线分析方法确定传导方向的波传播速度和色散。</li>
</ol>6. Förster 共振能量转移 (烦恼) 的循环腺苷磷酸 (阵营) 成像
注: 对于基于焦虑的测量, 收获心脏从 CAG-Epac1-camps 转基因小鼠16。
<ol><li>在显微镜15、17周围使用自构建的成像系统。</li>
	<li>把显微镜放在心脏前面, 调整它的视力。</li>
	<li>激发带有光源的野营传感器 [例如, 使用单波长发光二极管 (440 nm)]。使用光束分配器将发射光分成供体和受体通道 (对于青色荧光蛋白/黄色荧光蛋白对, 使用565dcxr 分色镜和 D480/30 和 D535/40 发射过滤器)。</li>
	<li>通过在会议厅周围放置一个塑料包装, 确保温度稳定。</li>
	<li>使用一个科学的互补金属氧化物半导体 (sCMOS) 相机拍摄图像。与开源成像软件 (如微管理器) 协调光源和相机图像捕获。</li>
	<li>要启动图像获取, 请推送多维 Acq。按钮和设置一个时间推移, 每十年代获取一个图像, 与适当的曝光时间, 这取决于荧光信号的强度 (大约100毫秒)。</li>
	<li>使用以前描述和可用的联机烦恼和烦恼在线 2插件17将图像拆分为两个通道, 选择感兴趣的区域, 并监视比率跟踪。</li>
	<li>在采集过程中, 根据实验的性质, 灌注了含有不同物质的改性克雷布斯 Henseleit 溶液的心脏。</li>
	<li>在实验结束时, 通过推送停止按钮并保存图像堆栈来关闭获取。</li>
	<li>使用专门的分析软件对烦恼数据进行离线分析, 它可以将图像分割为供方和受体通道的两个相同的部分, 并且可以在多个感兴趣的区域进行焦虑分析。 注意: 需要一个专用的插件 (烦恼脱机), 这是在 Sprenger et . 中提供的。17。<ol>
<li>启动软件。通过进入插件菜单, 然后单击事必躬亲, 然后在打开微管理器文件打开分析。</li>
		<li>运行烦恼脱机插件, 以便将时间间隔分割为 CFP 和 YFP 通道的两个相同的图像。</li>
		<li>使用手绘选项工具可以标记 YFP 图像中感兴趣的区域。按添加按钮将所选内容添加到多度量值窗口中。</li>
		<li>在多值窗口中选择感兴趣的区域, 然后按多以获取每个帧和区域的平均灰度值的表。将所有数据复制并粘贴到 Excel 工作表中。</li>
		<li>单击CFP 图像堆栈。执行与步骤6.10.4 中的 CFP 堆栈相同的操作, 并将平均灰色值粘贴到同一 Excel 工作表中。</li>
	</ol>
</li>
	<li>在接受方通道17中, 为捐赠者的出血通过因子脱机更正原始数据。<ol>
<li>使用以下公式, 其中 B 为出血系数: 比值 = (YFP x CFP)/CFP</li>
		<li>通过成像心脏仅表达 CFP 并测量 YFP 通道中供体荧光的百分比 (b = YFP/CFP) 确定出血通过因子 b。</li>
	</ol>
</li>
</ol>7. Neuromorphology
注: 采用完整的小鼠心脏 immunostainings, 分析心脏自主神经系统。注意, 大部分心内神经节是局部的在心外膜脂肪组织接近肺静脉。
<ol><li>使用不同的染色描述丝 (一般神经元结构; 鸡抗 NF-H, 1:3, 000), 酪氨酸羟化酶 (TH, 交感神经结构; 兔α TH, 1:1, 000), 胆碱乙酰转移酶 (聊天, 副交感神经神经元结构;山羊α聊天, 1:50)。</li>
	<li>在 Langendorff 装置中灌注后, 将10毫升福尔马林中的小鼠心脏固定24小时, 并将其储存在磷酸盐缓冲盐水 (PBS) 中, 4 ˚C。</li>
	<li>漂白的心脏在凹痕的漂白剂 (4:1: 1 甲醇: 过氧化氢溶液 30% (w/w) 在 H2O: 二甲基亚砜 (亚砜)) 1 星期在4˚C 并且水化他们随后对 pbs 在一系列下降的甲醇在 pbs (100%, 75%, 50%, 25%; 1 H 每个)18。</li>
	<li>以24井板格式执行以下孵化, 并在4˚C 中进行温和搅拌:<ol>
<li>Permeabilize 的心脏在 1% Triton-X-100/PBS (PBS t) 为 3x 1 小时在室温下阻断他们过夜在阻塞缓冲 [5% 牛血清白蛋白 (BSA)/PBS-T + 0.2% 叠氮化钠]。</li>
		<li>稀释抗体如下: 山羊α聊天 (1:50), 兔子α TH (1:1, 000), 鸡α丝 (1:3, 000);荧光标签的二级抗体 (1:500; 或根据制造商的指示);生物素化二级抗体的显色标签 (1:200; 或根据制造商的指示)。</li>
	</ol>
</li>
	<li>在4˚C 1 周, 在阻塞缓冲液中将原抗体中的标本孵育。</li>
	<li>在4天内, 在阻断缓冲器中, 在第二抗体孵化前, 将心脏洗净 3 x 15 分钟。</li>
	<li>在 PBS 中清洗 3 x 15 分钟的心脏, 并将它们存储在室温下进行荧光染色的安装介质中, 或者根据制造商的说明使用亲和生物素复合检测试剂盒。</li>
	<li>在一个商业缓冲器中预孵化1小时的心脏, 用于 33 '-diaminobenzidine (民建联), 然后根据制造商的指示, 在包含有缓冲的缓冲器的视觉控制下开发它们。</li>
	<li>将标本存放在双蒸馏水中。</li>
	<li>石蜡切片, 脱水和嵌入的心脏石蜡。<ol>
<li>切割4µm 厚的部分, 并根据实验室的例行程序 deparaffinize。是否和如何进行抗原检索需要建立每个单独的设置, 因为它取决于抗体组合。</li>
		<li>Permeabilize 10 分钟 0.2% Triton-X-100/Tris-buffered 盐水 (tb), 其次是 3 X 5 分钟洗涤在 tb。</li>
		<li>在室温下, 用 3% BSA/tb 的1小时阻挡它们。</li>
		<li>一夜之间孵化4˚C [主要抗体: 山羊α聊天 (1:50), 兔子α TH (1:500), 鸡α丝 (1:1, 000)] 或 2 h 在室温下 (荧光标记的二级抗体, 1:500) 在 1% BSA 或 tb 与 3 x 5 分钟洗涤之间的 tb。</li>
		<li>添加1µg/毫升的 bisbenzimide H33342 三盐酸盐二次抗体溶液或使用不同的核染色方法。</li>
		<li>将幻灯片安装在安装介质中进行荧光染色。</li>
	</ol>
</li>
</ol>

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W., 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=Enhanced+heterogeneity+of+myocardial+conduction+and+severe+cardiac+electrical+instability+in+annexin+A7-deficient+mice.">Enhanced heterogeneity of myocardial conduction and severe cardiac electrical instability in annexin A7-deficient mice.</a> Cardiovascular Research. 76 (2), 257-268 (2007).</li><li>Rudolph, V., 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=Myeloperoxidase+acts+as+a+profibrotic+mediator+of+atrial+fibrillation.">Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation.</a> Nature Medicine. 16 (4), 470-474 (2010).</li><li>Jungen, C., 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=Disruption+of+cardiac+cholinergic+neurons+enhances+susceptibility+to+ventricular+arrhythmias.">Disruption of cardiac cholinergic neurons enhances susceptibility to ventricular arrhythmias.</a> Nature Communications. 8, 14155 (2017).</li><li>Calebiro, D., 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=Persistent+cAMP-signals+triggered+by+internalized+G-protein-coupled+receptors.">Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.</a> PLoS Biology. 7 (8), e1000172 (2009).</li><li>Sprenger, J. U., Perera, R. K., G&#246;tz, K. R., Nikolaev, V. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+microscopy+for+real-time+monitoring+of+signaling+events+in+live+cells+using+unimolecular+biosensors.">FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors.</a> Journal of Visualized Experiments. (66), e4081 (2012).</li><li>Alanentalo, T., 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=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nature Methods. 4 (1), 31-33 (2007).</li><li>Whittington, N. C., Wray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+red+blood+cell+autofluorescence+for+immunocytochemistry+on+fixed+embryonic+mouse+tissue.">Suppression of red blood cell autofluorescence for immunocytochemistry on fixed embryonic mouse tissue.</a> Current Protocols in Neuroscience. 81, 2.28.1-2.28.12 (2017).</li><li>Fukuda, K., Kanazawa, H., Aizawa, Y., Ardell, J. L., Shivkumar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+innervation+and+sudden+cardiac+death.">Cardiac innervation and sudden cardiac death.</a> Circulation Research. 116 (12), 2005-2019 (2015).</li><li>Wengrowski, A. M., Wang, X., Tapa, S., Posnack, N. G., Mendelowitz, D., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+release+of+norepinephrine+from+cardiac+sympathetic+neurons+alters+mechanical+and+electrical+function.">Optogenetic release of norepinephrine from cardiac sympathetic neurons alters mechanical and electrical function.</a> Cardiovascular Research. 105 (2), 143-150 (2015).</li><li>Rivinius, R., 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=Control+of+cardiac+chronotropic+function+in+patients+after+heart+transplantation:+effects+of+ivabradine+and+metoprolol+succinate+on+resting+heart+rate+in+the+denervated+heart.">Control of cardiac chronotropic function in patients after heart transplantation: effects of ivabradine and metoprolol succinate on resting heart rate in the denervated heart.</a> Clinical Research in Cardiology. , (2017).</li><li>Ajijola, O. 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=Augmentation+of+cardiac+sympathetic+tone+by+percutaneous+low-level+stellate+ganglion+stimulation+in+humans:+a+feasibility+study.">Augmentation of cardiac sympathetic tone by percutaneous low-level stellate ganglion stimulation in humans: a feasibility study.</a> Physiological Reports. 3 (3), e12328 (2015).</li></ol>

作者没有什么可透露的。

本论文通过不同的制图和刺激技术, 提出了 Langendorff 的体外心脏灌注系统作为研究心内神经元对心脏电生理和心脏交感神经的影响的工具. 包括心内膜和心外膜的方法。
协议的几个部分对于安装来说是至关重要的。首先, 重要的是使用一种制备技术, 其中心房脂肪垫保持完好或迅速删除, 而不伤害心肌。第二, 适当大小的开放必须削减在右心房, 以方便插入 octapolar 导管进...

Langendorff 体外心脏灌注系统继续是一个相关的工具, 以执行广泛的生理, 生物化学, 形态学和药理学研究中枢失神经心脏 1,2 ,3,4,5自其发明在后期 19th世纪6。迄今为止, 该系统仍然广泛用于各种主题 (e. g, 缺血再灌注) 或研究心脏药理作用7,8, 是心血管研究的基本工具。这种方法的长寿源于几个优点 (e. g), 测量结果不受中枢神经系统或其他器官、系统循环或循环激素的影响。如有需要, 可以以控制方式将药物添加到灌注缓冲器或直接应用于特定结构。实验是可重现的, 在很短的时间内可以进行比较高的实验。(部分) 开放性质的设置可以使温度调节困难, 需要考虑。虽然 Langendorff 系统也用于较大的物种9, 但较小的动物主要用于实验性的设置不太复杂, 更大的生物变异性 (g., 转基因小鼠模型) 可以使用。
在本协议的实验设置中, 心内自主神经系统对基本电生理参数、心室心脏交感神经、心外膜传导、环腺苷 (阵营) 动力学的影响是评价。大量的心内神经节, 主要位于心房脂肪垫, 现在众所周知的控制心脏电生理独立于中枢神经控制, 要么保持完好或手动删除与仔细机械夹层。对自主神经系统的药理调节, 要么通过向灌注缓冲器添加药物, 要么在局部通过心房神经节的靶向调节来进行。实验后, 心脏是非常适合的免疫组织化学评估, 因为所有的血细胞已被删除由于连续灌注, 这可以提高染色质量。
所述技术的总体目标是为详细研究自主神经系统对心脏电生理和心脏交感神经的影响提供新的视角。使用这种技术的一个原因是, 在没有中枢神经系统影响的情况下, 可以研究和改变自主神经系统。一个主要的好处是容易的就业的药理实验, 其中潜在的亲或抗心律失常性质的新旧的药物可以测试。此外, 各种心脏疾病的转基因和击倒老鼠模型可以用来研究心律失常、心力衰竭或代谢性疾病的机制。这种方法增强了我们对心房的自主神经系统对心室心脏电生理的影响以及心律失常的诱导的理解。

<table><tbody><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Sodium hydrogencarbonate</td><td>Sigma-Aldrich</td><td>401676</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5655</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>M1880</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Sodium pyruvate bioXtra</td><td>Sigma-Aldrich</td><td>P8574</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Carbogen (95% O&lt;sub&gt;2&lt;/sub&gt; / 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany</td><td>Modified Krebs-Henleit solution</td><td></td></tr><tr><td>Sterile filter steritop-GP 0.22</td><td>EMD Millipore</td><td>SCGPT05RE</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Atropine sulfate</td><td>Sigma-Aldrich</td><td>A0257</td><td>Neuromodulation</td></tr><tr><td>Hexamethonium chloride</td><td>Sigma-Aldrich</td><td>H2138</td><td>Neuromodulation</td></tr><tr><td>Nicotine free base 98-100%</td><td>Sigma-Aldrich</td><td>N3876</td><td>Neuromodulation</td></tr><tr><td>Formalin solution neutral buffered 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Whole mount staining</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859</td><td>Whole mount staining</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>Whole mount staining</td></tr><tr><td>Hydrogen peroxide solution 30% (w/w) in H2O</td><td>Merck, KGA, Darmstadt, Germany</td><td>H1009</td><td>Whole mount staining</td></tr><tr><td>Dimethyl sulfoxide</td><td>Merck, KGA, Darmstadt, Germany</td><td>D8418</td><td>Whole mount staining</td></tr><tr><td>Phosphate-buffered saline tablets</td><td>Gibco / Invitrogen</td><td>18912-014</td><td>Whole mount staining</td></tr><tr><td>Triton-x-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Whole mount staining</td></tr><tr><td>Albumin bovine fraction V</td><td>Biomol, Hamburg, Germany</td><td>11924.03</td><td>Whole mount staining</td></tr><tr><td>Chicken anti neurofilament</td><td>EMD Millipore</td><td>AB5539</td><td>Whole mount staining</td></tr><tr><td>Rabbit anti tyrosine hydroxylase</td><td>EMD Millipore</td><td>AB152</td><td>Whole mount staining</td></tr><tr><td>Goat anti choline acetyltransferase</td><td>EMD Millipore</td><td>AP144P</td><td>Whole mount staining</td></tr><tr><td>Donkey &amp;alpha; rabbit IgG Alexa 488</td><td>Thermo Fisher Scientific</td><td>A21206</td><td>Whole mount staining</td></tr><tr><td>Donkey &amp;alpha; goat IgG Alexa 568</td><td>Thermo Fisher Scientific</td><td>A11057</td><td>Whole mount staining</td></tr><tr><td>Donkey &amp;alpha; chicken IgY Alexa 647</td><td>Merck, KGA, Darmstadt, Germany</td><td>AP194SA6</td><td>Whole mount staining</td></tr><tr><td>Biotin-conjugated donkey &amp;alpha; rabbit igG</td><td>R&amp;amp;D Systems</td><td>AP182B</td><td>Whole mount staining</td></tr><tr><td>Biotin-conjugated donkey &amp;alpha; goat igG</td><td>R&amp;amp;D Systems</td><td>AP192P</td><td>Whole mount staining</td></tr><tr><td>Biotin-conjugated goat &amp;alpha; chicken igY</td><td>R&amp;amp;D Systems</td><td>BAD010</td><td>Whole mount staining</td></tr><tr><td>Vectashield mounting medium</td><td>Vector laboratories, Burlingame, CA, USA</td><td>H-1000</td><td>Immunohistochemistry</td></tr><tr><td>Vectastain ABC kit</td><td>Vector laboratories, Burlingame, CA, USA</td><td>PK-4000</td><td>Immunohistochemistry</td></tr><tr><td>Steady DAB/Plus</td><td>Abcam plc, Cambridge, UK</td><td>ab103723</td><td>Whole mount staining</td></tr><tr><td>HistoClear</td><td>DiaTec, Bamberg, Germany</td><td>HS2002</td><td>Immunohistochemistry</td></tr><tr><td>BisBenzimide H33342 trihydrochloride (Hoechst)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>B2261</td><td>Immunohistochemistry</td></tr><tr><td>Vectashield HardSet mounting medium</td><td>Vector laboratories, Burlingame, CA, USA</td><td>VEC-H-1400</td><td>Immunohistochemistry</td></tr><tr><td>Perfusion system</td><td>HUGO SACHS ELEKTRONIK - HARVARD APPARATUS GmbH, March-Hugstetten, Germany</td><td>&amp;nbsp;73-4343</td><td>Langendorff apparatus</td></tr><tr><td>Data acquisition system and corresponding software for catheter and physiological parameter</td><td>Powerlab 8/30 &amp;amp; Labchart, ADInstruments, Dunedin, New Zealand</td><td>PL3508 PowerLab 8/35</td><td>Langendorff setup</td></tr><tr><td>Octapolar catheter</td><td>CIB&amp;rsquo;ER Mouse, NuMed Inc., Hopkinton, NY, USA</td><td>custom</td><td>Langendorff setup</td></tr><tr><td>Stimulus generator</td><td>STG4002, Multi Channel Systems, Reutlingen, Germany</td><td>STG4002-160&amp;micro;A</td><td>Stimulation setup</td></tr><tr><td>Stimulation software</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>MC_Stimulus II</td><td>Stimulation setup</td></tr><tr><td>Data acquisition system and corresponding software for epicardial electrograms</td><td>ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany</td><td>USB-ME128-System</td><td>MEA setup</td></tr><tr><td>Multi-electrode array</td><td>MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany</td><td>EcoFlexMEA36</td><td>MEA setup</td></tr><tr><td>Multi-electrode array recording software</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>MC_Rack</td><td>MEA setup</td></tr><tr><td>Spring scissors</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>15003-08</td><td>Heart Preparation</td></tr><tr><td>Strabismus Scissors</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>14575-09</td><td>Heart Preparation</td></tr><tr><td>Mayo Scissors</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>14110-15</td><td>Heart Preparation</td></tr><tr><td>Dumont SS Forceps</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>11203-25</td><td>Heart Preparation</td></tr><tr><td>London Forceps</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>11080-02</td><td>Heart Preparation</td></tr><tr><td>Narrow Pattern Forceps</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>11003-13</td><td>Heart Preparation</td></tr><tr><td>Plastic Wrap</td><td>Parafilm M, Bemis NA, based in Neenah, WI, United States</td><td></td><td>Consumable Materials</td></tr><tr><td>Stereomicroscope</td><td>Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany</td><td></td><td>FRET</td></tr><tr><td>LED</td><td>CoolLED, Andover, UK</td><td>&lt;em&gt;p&lt;/em&gt;E-100</td><td>FRET</td></tr><tr><td>DualView</td><td>Photometrics, Tucson, AZ, USA</td><td>DV2-SYS</td><td>FRET</td></tr><tr><td>DualView filter set</td><td>Photometrics, Tucson, AZ, USA</td><td>05-EM</td><td>FRET</td></tr><tr><td>optiMOS scientific CMOS camera</td><td>Qimaging, Surrey, BC, Canada</td><td>01-OPTIMOS-R-M-16-C</td><td>FRET</td></tr><tr><td>Imaging software &amp;nbsp;</td><td>Micro-Manager; Vale Lab, University of California San Francisco, CA, USA</td><td></td><td>FRET</td></tr><tr><td>Analysis Software</td><td>Image J software; Public Domain, NIH, USA</td><td></td><td>FRET</td></tr></tbody></table>

impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo langendorff system

自19晚期th世纪的发明以来, Langendorff前体心脏灌注系统继续是研究广泛的生理、生物化学、形态学和药理学参数的相关工具。中枢失神心。在这里, 我们描述了调节心脏内自主神经系统的设置和评估其对基本电生理, 心脏交感神经和循环腺苷 (阵营) 动力学的影响。心内自主神经系统由心房脂肪垫的机械解剖调节--小鼠神经节主要位于--或使用全球以及靶向的药理干预。将 octapolar 电生理导管引入右心房和右心室, 用心外膜放置的多电极阵列进行高分辨率映射, 以确定心脏电生理和心脏交感神经。Förster 共振能量转移 (焦虑) 成像进行实时监测的营地水平在不同的心脏地区。采用神经标记法对 Neuromorphology 的全心抗体染色方法进行研究, 以指导心脏自主神经系统特定靶点的识别和调制。前体 Langendorff 设置允许在短时间内进行大量的可重现实验。然而, 设置的部分开放性质 (e. g) 使恒温控制变得困难, 应保持在最低限度。该方法可以对分散心内心脏自主神经系统进行分析和调节。

图 1显示了 Langendorff 设置的映像, 其中包括2个多电极阵列 (多边环境协定)。实验前, 心内导管定位在套管附近, 方便快速方便地插入右心房/右心室, 确保在平衡开始之前有很短的时间。房间的下部可以提高 (请参见图 1中的箭头), 以使会议厅完全闭合并保证稳定的温度。
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Watch this Scientific Journal Video about Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System at JoVE.com

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

在这里, 我们提出了一个调节心内自主神经系统的协议, 并评估其对基本的电生理, 心脏交感神经和阵营动力学的影响使用前体 Langendorff 设置.

心内神经元对体外 Langendorff 系统心脏电生理和心脏交感神经的影响

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

impact-intracardiac-neurons-on-cardiac-electrophysiology

Research

JoVE Journal

Medicine

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

9.8K 次观看.  University Hospital Hamburg-Eppendorf. 在这里, 我们提出了一个调节心内自主神经系统的协议, 并评估其对基本的电生理, 心脏交感神经和阵营动力学的影响使用前体 Langendorff 设置. 

视频: Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

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 ...

科研

生物工程

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.

前维沃·兰根多夫注入心脏的心电生理学研究

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

医学

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

生物学

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

微电极阵列记录中天节点发射率，以识别小鼠内在心脏起搏缺陷

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

新生儿鼠心在朗根多夫制剂中的应用改良技术

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia.
Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering.
A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

The combination of laser poration and microelectrode arrays (MEA) allows action potential-like recordings of cultivated primary and stem cell-derived cardiomyocytes. The waveform shape provides superior insight into test compounds' mode of action than standard recordings. It links patch-clamp and MEA readout to further optimize cardio safety research in the future.

激光诱导动作电位样测量微电极阵列上的心肌细胞，以提高安全药理学的预测性

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small ...

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

啮齿动物心脏中的病毒转基因表达和心律失常风险的评估

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

Optogenetic Activation of Intrinsic Cardiac Autonomic Neurons in Excised Perfused Mouse Hearts

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can be done using optogenetics through selective expression of channelrhodopsin-2 (ChR2) in cardiac autonomic neurons. Such cardiac applications of optogenetics allow for the study of the intrinsic release of neurotransmitters in a spatiotemporal manner. This method illustrates an ex vivo approach for specific optogenetic stimulation of cardiac neurons in perfused mouse hearts. Transgenic mice were bred to express ChR2 in either choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) neurons throughout the body. A micro-LED (465 nm) encased in a silicone elastomer was prepared for stimulating the neurons of the right atrium that innervate the sinoatrial node. The micro-LED was connected to a function generator set to pulse waves at 10 Hz with a 30 ms pulse width. Hearts with confirmed expression were excised and retrogradely perfused on a Langendorff system circulating Krebs-Henseleit solution. Electrocardiogram (ECG), temperature, and coronary flow rate were recorded using the LabChart software. Once the heart stabilized, the micro-LED was placed on the right atrium and tested for optimal heart rate response. An application of this approach combines the intrinsic release of cholinergic neurotransmitter (acetylcholine) during optogenetic activation of a ChAT-ChR2 mouse heart simultaneously with increasing exogenous catecholaminergic neurotransmitter (norepinephrine) added to the perfusate. The resulting changes in heart rate during the simultaneous cholinergic and catecholaminergic activation are presented. This method describes a valuable experimental approach for investigating the kinetics of sudden intrinsic autonomic neuron activation in perfused hearts and the interactions between cardiac cholinergic and catecholaminergic activity.

This protocol illustrates a method for optogenetic stimulation of intrinsic cardiac neurons in transgenic mouse hearts. The approach described is used for investigating the kinetics of sudden cardiac neuron activation in ex vivo perfused hearts and the interactions between cholinergic and catecholaminergic activity.

切除灌注小鼠心脏中内源性心脏自主神经神经元的光遗传学激活

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can ...

从微观到宏观尺度的人类心脏综合 3D 分析

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results&#160;to illustrate progress in the research of human cardiac anatomy.

作者聚焦:通过心脏多尺度分析推进人类心脏解剖学

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

用于人类心脏多尺度三维解剖学研究的管道

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic ...