我们感谢为教育和研究的进步而捐献遗体的个人。我们感谢 OneLegacy 基金会，它为获得供体心脏进行研究奠定了基础。我们还感谢加州大学洛杉矶分校转化研究成像中心（放射学系）的 Anthony A. Smithson 和 Arvin Roque-Verdeflor 在 CT 数据采集方面的支持。该项目得到了加州大学洛杉矶分校 Amara Yad 项目的支持。我们感谢 Kalyanam Shivkumar 博士和 Olujimi A. Ajijola 博士建立和维护人类心脏研究管道。我们感谢我们的研究运营经理 Amiksha S. Gandhi 对支持我们项目的奉献精神。这项工作得到了NIH赠款OT2OD023848和P01 HL164311和Leducq赠款23CVD04给Kalyanam Shivkumar的支持，美国心脏协会职业发展奖23CDA1039446给PH，以及UCLA Amara-Yad项目（https://www.uclahealth.org/medical-services/heart/arrhythmia/about-us/amara-yad-project 的支持。本研究中使用的 GNEXT microPET/CT 扫描仪由 NIH 动物研究资助共享仪器 （1 S10 OD026917-01A1） 资助。

这项研究使用了从非失败的供体人类心脏中收集的去识别化组织样本，并获得了加州大学洛杉矶分校 （UCLA） 机构审查委员会的批准。样本来自被拒绝移植的未衰竭心脏。对心脏进行压力灌注，用 4% 多聚甲醛 （PFA） 固定，并在组织处理前按照以下方法进行成像。 图 1 总结了研究顺序的流程图。材料 表中列出了研究中使用的试剂和设备的详细信息。
1. 微观检查
<ol><li>使用改良免疫标记的溶剂透明化器官 3D 成像 （iDISCO+） 方案进行组织透明化。<ol><li>用手术刀解剖 4% PFA 固定的组织，使其适合 3 mm × 16 mm × 25 mm 的共聚焦显微镜室。为了对较厚的组织进行成像，可以在载玻片上堆叠额外的腔室和/或垫片。</li>
		<li>使用分级甲醇 （MeOH） 系列（20%、40%、60%、80% 和 100% MeOH 在去离子 H2O [体积/体积]）下在室温 （RT） 下搅拌每个样品 1 小时。</li>
		<li>在 RT 下用 100% MeOH 洗涤 1 小时，然后在 RT 下浸入 66% 二氯甲烷/33% MeOH 中搅拌过夜。</li>
		<li>第二天，在 RT 下用 MeOH （100%） 洗涤两次 1 小时，在 4 °C 下冷却，并在 4 °C 下用 5% H2O2 在 MeOH （vol/vol） 中处理过夜。</li>
		<li>用分级的 MeOH 系列（80%、60%、40% 和 20% MeOH）再水化，并在 0.01 mol/L PBS 中洗涤 1 小时，每次在 RT 下搅拌。</li>
		<li>在 RT 下，在 0.01 mol/L PBS 和 0.2% Triton X-100 中洗涤组织两次，持续 1 小时。</li>
		<li>在 0.01 mol/L PBS、20% 二甲基亚砜 （DMSO）、0.2% Triton X-100 和 0.3 mol/L 甘氨酸中透化 2 天，在 37 °C 下搅拌，制备免疫标记。</li>
		<li>在 0.01 mol/L PBS 中，含 10% DMSO、0.2% Triton X-100 和 5% 正常驴血清，在 37 °C 下搅拌再封闭 2 天。</li>
		<li>使用与偶联荧光团的 MeOH 相容的一抗进行标记，该荧光团在 0.01 mol/L PBS 中稀释，其中含有 10 mg/mL 肝素 （PTwH）、0.2% 吐温-20、5% DMSO 和 3% 正常驴血清，在 37 °C 下搅拌 3-4 天。</li>
		<li>补充抗体溶液，并在 37 °C 下搅拌再孵育 3-4 天。</li>
		<li>在一抗溶液中孵育 1 周后，在 RT 下用 PTwH 洗涤 4 至 5 次过夜。</li>
		<li>与在 PTwH、3% 正常驴血清中稀释的荧光团偶联的二抗在 37 °C 下搅拌孵育 3 天。</li>
		<li>补充二抗溶液，在 37 °C 下搅拌再孵育 3 天。</li>
		<li>在二抗溶液中孵育 6 天后，在 RT 下用 PTwH 洗涤 4-5 次过夜。</li>
		<li>用分级的 MeOH 系列（20%、40%、60%、80%、100% 和 100% MeOH）脱水。样品可以在 RT 中储存过夜。</li>
		<li>在 RT 下在 66% 二氯甲烷/33% MeOH 中孵育 3 小时，同时搅拌。</li>
		<li>在 RT 下用 100% 二氯甲烷洗涤两次，每次 15 分钟，并搅拌。</li>
		<li>将标本孵育并储存在苄基醚中。填充试管以尽量减少空气氧化样品。</li>
	</ol></li>
	<li>对组织透明化标本进行成像<ol><li>将含有粘合剂的腔室固定在载玻片上，并在腔室周边涂抹指甲油。对于较厚的组织，可以在载玻片上堆叠额外的腔室和/或垫片。</li>
		<li>将透明化的组织放入腔室中，填充苄醚，并盖上盖玻片。</li>
		<li>在盖玻片周围涂上指甲油以形成密封。</li>
		<li>使用带有 5 倍或 10 倍镜头的正置激光扫描共聚焦显微镜获取 tilescan 和 Z 堆栈图像，以在镜头工作距离的深度成像。</li>
		<li>使用适用于所用荧光团发射光谱的激光线以 1024 x 1024 的分辨率成像。使用 488 nm 激光线可以看到肌肉自发荧光。</li>
		<li>确保 z 轴步长与基于指定物镜11 的数值孔径的奈奎斯特采样相称。</li>
		<li>拼接图像并使用软件进行 3D 可视化。</li>
		<li>使用单个和合并通道的 Z 堆栈的最大强度投影 （MIP） 图像创建图形（图 2）。</li>
	</ol></li>
	<li>免疫组化 注:在组织石蜡包埋22 后，使用以下程序创建用于免疫组织化学研究的载玻片。<ol><li>制备折射率匹配溶液 （RIMS）。<ol><li>向去离子 H2O 中加入 10.9 g Na2HPO4（无水）和 3.1 g NaH2PO4（一水合物），制备 0.1 mol/L 磷酸盐缓冲液，总体积为 1 L （pH 7.4）。过滤消毒溶液并将其储存在 RT 中。</li>
			<li>将磷酸盐缓冲液稀释至 0.02 mol/L。</li>
			<li>用磁力搅拌棒在最终储存瓶中搅拌溶液 10 分钟，将 Histodenz 溶解在 30 mL 0.02 mol/L 磷酸盐缓冲液中，该储存瓶可以密封以最大限度地减少蒸发和污染。</li>
			<li>加入叠氮化钠至总浓度为 0.01% （w/v），并用 NaOH 将 pH 值调节至 7.5。</li>
			<li>通过改变 Histodenz 的最终浓度来调整 RI。</li>
			<li>将 RIMS 在 RT 中存放数月。如果发现微生物污染，请丢弃。 注意:请勿高压灭菌任何含有叠氮化钠的溶液。</li>
		</ol></li>
		<li>制备用于免疫组织化学研究的载玻片<ol><li>用切片机创建 5 μm 厚的切片。将组织切片应用于带电的载玻片。</li>
			<li>通过将载玻片在 >75% 二甲苯中孵育 10 分钟来去除石蜡。将玻片移至装有二甲苯的第二个容器中再放置 10 分钟。</li>
			<li>将载玻片浸入 100% 乙醇中 10 分钟，然后在 95% 乙醇中浸泡 5 分钟，在 70% 乙醇中浸泡 5 分钟，去除二甲苯。</li>
			<li>用去离子 H2O 冲洗载玻片 5 分钟。</li>
			<li>将玻片在 90-95°C 下浸入抗原修复缓冲液中 25 分钟。</li>
			<li>让容器在搅拌下冷却至 RT 1 小时。</li>
			<li>将载玻片浸入浸泡缓冲液 （0.01 mol/L PBS + 0.4% Triton X-100） 中，在 4°C 下浸泡 30 分钟。</li>
			<li>用 PAP 笔圈住组织。将 PBS 添加到每张载玻片中，并将其放入加湿室中以防止干燥。</li>
			<li>在 RT 下用 PBS 洗涤载玻片，搅拌 5 分钟。</li>
			<li>用封闭缓冲液（0.01 mol/L PBS + 10% 驴血清 + 0.1% TX-100）封闭 1 小时，同时搅拌。</li>
			<li>与在封闭缓冲液中稀释的一抗在 4°C 下孵育过夜。</li>
			<li>第二天，让载玻片升温至 RT 15 分钟。</li>
			<li>用 0.01 mol/L PBS + 0.2% TritonX-100 洗涤载玻片 3 次，每次 5 分钟。</li>
			<li>与在封闭缓冲液中稀释的二抗在 RT 下搅拌孵育 1 小时。</li>
			<li>用 0.01 mol/L PBS + 0.2% TritonX-100 洗涤载玻片 3 次，每次 5 分钟。</li>
			<li>用 0.01 mol/L PBS 洗涤载玻片 3 次，每次 5 分钟。</li>
			<li>用滴管滴入 1 滴 RIMS 并盖上盖玻片。</li>
			<li>在盖玻片周围涂上指甲油以形成密封。</li>
			<li>作为阴性对照，在没有一抗的情况下运行样品，以证明不存在特异性染色。</li>
		</ol></li>
		<li>免疫染色玻片成像<ol><li>使用带有 10 倍、20 倍和 40 倍物镜的激光扫描共聚焦显微镜对载玻片进行成像。</li>
			<li>使用适合所用二抗发射光谱的激光线以 1024 x 1024 的分辨率成像。</li>
			<li>使用单个和合并通道的 Z 堆栈的最大强度投影 （MIP） 图像创建图形（图 3）。</li>
		</ol></li>
	</ol></li>
</ol>2. 中尺度检查
<ol><li>立体解剖<ol><li>使用带夹的放大台灯、手术望远镜或体视显微镜，对微小或薄的结构进行精细解剖，例如房室结、房室结动脉和心脏神经丛（亚毫米到毫米级）。</li>
	</ol></li>
	<li>显微 CT 扫描 注意:在压力灌注和固定后以及使用微正电子发射断层扫描 （PET）/CT 扫描仪在解剖的任何阶段进行 CT 成像（图 4）。<ol><li>在样品成像之前，将 CT X 射线源预热 25 分钟。</li>
		<li>将心脏样本放在扫描仪床上。</li>
		<li>将扫描仪床移动到 544 mm 的水平位置和 14 mm 的垂直位置，以使心脏在 CT 视野 （FOV） 中居中。</li>
		<li>以 80 kVp、150 μA 的速度获取 CT 图像，在 1 分钟的扫描时间内以 200 μm 的空间分辨率进行 720 次投影。</li>
		<li>使用 12 厘米 x 12 厘米 x 10 厘米的视野和 600 x 600 x 500 体素的矩阵重建 CT 数据，并另存为 DICOM 文件。</li>
	</ol></li>
</ol>3. 宏观考察
<ol><li>加压灌注和固定 注:作者修改了先前描述的压力灌注和固定技术，并将其应用于拒绝移植的非失败人类心脏 14,19,20,21。<ol><li>使用高流量泵进行灌注固定。使用 100% 乙醇14、4% PFA 或 10% 福尔马林作为固定剂。 注意:通过尽可能远端切除升主动脉、肺干、腔静脉和肺静脉来恢复心脏，并在威斯康星大学解决方案23 中输送。</li>
		<li>使用两个 20-24 Fr 手术插管进行右心和左心灌注。对于右心灌注，插管上腔静脉，并用半切 12-30 mL 大小的塑料注射器在肺干或肺动脉处放置一个通气口，该注射器带有连接到三通旋塞阀的 Luer-Lock 尖端。</li>
		<li>放入适当大小的锁定半切塑料注射器或 1.5-5.0 mL 离心管后，用麻线封闭下腔静脉和另一条肺动脉。<ol><li>对于左心的顺行灌注，将其中一条肺静脉插管，并使用半切 12-30 mL 大小的塑料注射器在主动脉的远端放置一个通风口，该注射器带有连接到三通旋塞阀的 Luer-Lock 尖端。</li>
			<li>对于左心的逆行灌注，将其中一个主动脉弓分支插管，并在主动脉弓的另一个分支处放置一个通风口。将插管的尖端放在两个心室中。</li>
		</ol></li>
		<li>插入适当大小的锁定半切塑料注射器或 1.5-5.0 mL 微量离心管后，用麻线堵塞其他血管孔。用薄纱布盖住注射器/管子/插管的插入部分，以防止渗漏和滑落。使用缝合、绑扎或结块修复大渗漏。允许少量泄漏。</li>
		<li>将心脏悬在塑料容器中。</li>
		<li>将 22-24 Fr 软塑料管连接到每个套管上，并将管子的另一端插入装满固定剂的容器中。</li>
		<li>使用高流量泵在右心和左心回路中循环固定剂，右心和左心分别达到约 100-300 mL/min 和 200-400 mL/min，分别在右心室达到约 20 mmHg 和左心室约 80 mmHg。</li>
		<li>在 4 °C 维持灌注 24 小时。</li>
		<li>用 0.01 mol/L PBS 洗涤心脏 30 分钟，搅拌 4 次。</li>
		<li>将心脏储存在 4 °C 的 0.01 mol/L PBS/0.02% 叠氮化钠中。 注意:压力灌注固定仅对新鲜心脏有效，对从防腐尸体中恢复的心脏无效。</li>
	</ol></li>
	<li>大体解剖<ol><li>在解剖的每个阶段使用照片记录进行渐进式解剖。</li>
		<li>为了保持临床相关性，请特别注意避免扭曲/变形任何结构，以维持心脏的生理形态。</li>
		<li>使用临床相关方向（例如右前斜方向）对目标结构进行成像。</li>
	</ol></li>
	<li>摄影<ol><li>将压力灌注和固定的心脏放在三脚架上，该平台安装有多个叉子，并能够 360度旋转。</li>
		<li>使用数码单镜头反光相机（图 5）24 拍摄心脏，同时使用 C 型支架上的多个发光二极管灯板和宽大的黑色羽绒被背景布。</li>
		<li>使用长焦距 （200 mm） 镜头拍摄 4-6 英尺的工作距离，以最大限度地减少拍摄对象的失真14.</li>
	</ol></li>
	<li>浮雕<ol><li>要显示立体图像，请从 CT 数据集重建一对照片或体积渲染图像，在水平面上的旋转角度相差 10°。</li>
		<li>使用免费软件16 将这些二维 （2D） 图像（称为立体图）转换为立体图。</li>
		<li>要查看立体图，请使用红色/青色眼镜。</li>
	</ol></li>
	<li>摄影 测量 注:摄影测量是从以不同角度拍摄的多张二维照片中生成三维表面渲染重建的应用科学17。<ol><li>将样品悬挂在 C 型支架上或将其放在旋转台上，以使用智能手机获得数百张多向照片。</li>
		<li>使用市售软件以 FBX 格式生成 3D 模型。</li>
	</ol></li>
	<li>CT 扫描 注意:CT 扫描可以在压力灌注和固定后以及解剖的任何阶段进行。<ol><li>将心脏样本悬浮在容器顶部的杆上。为防止心脏在扫描过程中摆动，请用固定在容器底部的塑料尖头支撑心脏底部。因此，空气将起到负对比的作用。</li>
		<li>使用市售的多探测器行 CT 扫描仪进行 CT 扫描，参数如下:管电压为 120 kV，管电流为 800-900 mA，机架旋转为 280 ms。剂量长度产品一般为 500-1200 mGy.cm。</li>
		<li>使用以下参数重建轴向图像数据:截面厚度，0.6 毫米;增量间隔，0.3 毫米;视野，尽可能小（通常为 100-200 毫米）;和一个矩阵，512 × 512。</li>
	</ol></li>
	<li>虚拟解剖<ol><li>使用市售软件分析 CT 扫描图像以生成虚拟解剖图像。 注意:虚拟解剖是体积渲染过程的修改，其中焦点转移到心腔和血管壁18。在此过程中，手动阈值实际上从原始数据集中删除了增强的腔室。</li>
		<li>通过虚拟解剖可视化非增强的壁、隔膜和瓣膜，以生成类似于大体解剖的图像。与心脏标本的粗略解剖不同，虚拟解剖过程中的切割平面实际上是无限的。几乎任何视图都可以根据需要重新创建以可视化感兴趣的结构。</li>
	</ol></li>
	<li>3D 打印<ol><li>在 3D 打印机软件中打开心脏样本的兼容文件。</li>
		<li>在 3D 打印机的打印设置中使用 0.10 mm Fast DETAIL 的配置文件，并将打印速度降低到 20 mm/s。启用 Generate support material（生成支持材料）。</li>
		<li>在 3D 打印机中将 TPU 线材的配置文件 用于"线材设置"。</li>
		<li>将 Original Prusa MK4 Input Shaper 0.4 喷嘴 的配置文件用于打印机中的"打印机设置"。</li>
		<li>切片完成后，将 BGCODE 文件保存在 USB 闪存驱动器中进行 3D 打印。</li>
		<li>使用 1.75 毫米 TPU 细丝进行 3D 打印人体心脏样本。在 3D 打印之前，使用细丝干燥器将 TPU 细丝干燥 6 小时。</li>
		<li>为了减少 3D 打印过程中的线丝张力，请将线轴放在带有内置轴承的线轴支架上，以方便线轴旋转。使用市售的 3D 打印机和纹理粉末涂层钢板进行 3D 打印。</li>
		<li>3D 打印完成后，小心地去除支撑材料。</li>
	</ol></li>
</ol>

从微观到宏观尺度的人类心脏综合 3D 分析

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本研究展示了分析从全人类心脏获得的样本的综合管道。代表性结果显示，对单个心脏进行常规的微观到宏观解剖检查。由于人类心脏样本非常珍贵，因此多尺度方法是理想且有效的，这样就不会通过为各种目的应用多种方案来浪费样本的任何部分，包括科学研究中的发现、临床程序的改进和解剖学教育，同时保持解剖学相关性在整个心脏的背景下。
关于微量检查，免疫染?...

由于功能服从形式，了解心脏的结构是欣赏其生理学的基础。尽管大量研究揭示了从微观到宏观的心脏解剖结构 1,2,3，但多个问题仍未解决，尤其是与人类心脏解剖学相关的问题。这部分是因为专注于功能解剖学的基础研究通常使用动物心脏 4,5,6，这通常与人类心脏 1,7,8 不同。此外，每项单独的研究，即使是那些使用人类心脏样本的研究，也往往侧重于非常具体的结构，这使得很难在整个心脏的背景下应用这些发现。如果聚焦结构位于微尺度或中尺度，例如 perinexus9 和 ganglionated plexus10，则更是如此。
在这种情况下，对被拒绝移植的人类心脏的系统结构研究提供了一个独特而难得的机会，可以获得微观和宏观尺度上聚焦的心脏结构的综合图谱11。显微镜检查方案包括组织透明化（溶剂透明化器官的改良免疫标记启用三维 （3D） 成像，iDISCO+）12,13 和免疫组织化学染色。中镜检查方案包括立体解剖、宏摄影和显微计算机断层扫描 （CT）。肉眼检查方案包括大体解剖14、摄影（包括立体画和摄影测量）15、16、17、CT、虚拟解剖18 和物理或虚拟解剖或整个心脏的 3D 打印17。为了准备肉眼检查，进行压力灌注固定以保持心脏的 3D 结构和生理相关形态 14,19,20,21。这些技术的组合应用是独一无二的，对于在人体心脏的 3D 结构背景下关联不同的解剖特征至关重要。
由于获得非病理性人类心脏样本的机会极其有限，因此本文描述的多尺度方法可以最大限度地利用样本。通过应用下面描述的各种程序，具有代表性的结果将向读者说明如何将这些发现用于多种目的，包括科学研究11 中的发现（心脏神经支配的综合分析、神经节神经丛的分布）、临床程序的改进（手术和介入方法的模拟）和解剖学教育（心脏解剖学的真实 3D 演示）。

<table><tbody><tr><td>1x Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P3813</td><td></td></tr><tr><td>3D Viewer</td><td>Microsoft</td><td></td><td></td></tr><tr><td>647 AffiniPure Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>711-605-152</td><td></td></tr><tr><td>647 AffiniPure Donkey Anti-Sheep IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>713-605-147</td><td></td></tr><tr><td>AF Micro-NIKKOR 200 mm f/4D IF-ED lens</td><td>Nikon</td><td></td><td></td></tr><tr><td>Anti-Actin, &amp;alpha;-Smooth Muscle - Cy3 antibody</td><td>Sigma-Aldrich</td><td>C6198</td><td></td></tr><tr><td>Antigen Retrieval Buffer (100x EDTA Buffer, pH 8.0)</td><td>Abcam</td><td>ab93680</td><td></td></tr><tr><td>Anti-PGP9.5 (protein gene product 9.5)</td><td>Abcam</td><td>ab108986</td><td></td></tr><tr><td>Anti-TH (tyrosine hydrox ylase)</td><td>Abcam</td><td>ab1542</td><td></td></tr><tr><td>Anti-VAChT (vesicular acetylcholine transporter)</td><td>Synaptic Systems</td><td>139 103</td><td></td></tr><tr><td>Benzyl ether</td><td>Sigma-Aldrich</td><td>108014</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4503-10G</td><td></td></tr><tr><td>Cheetah 3D printer filament (95A), 1.75 mm</td><td>NinjaTek</td><td></td><td></td></tr><tr><td>Coverslip, 22 mm x 30mm, No. 1.5</td><td>VWR</td><td>48393 151</td><td></td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>711-165-152</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich</td><td>270997-100ML</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418-500ML</td><td></td></tr><tr><td>Ethanol, 100%</td><td>Decon laboratories</td><td>2701</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126-500G</td><td></td></tr><tr><td>GNEXT PET/CT</td><td>SOFIE Biosciences</td><td></td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma-Aldrich</td><td>H3149-50KU</td><td></td></tr><tr><td>Histodenz</td><td>Sigma-Aldrich</td><td>D2158-100G</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>H1009-500ML</td><td></td></tr><tr><td>Imaging software</td><td>Zeiss</td><td>ZEN (black edition)</td><td></td></tr><tr><td>Imaging software</td><td>Oxford Instruments</td><td>Imaris 10</td><td></td></tr><tr><td>iSpacer</td><td>Sunjin Labs</td><td>iSpacer 3mm</td><td></td></tr><tr><td>KIRI Engine</td><td>KIRI Innovation</td><td></td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>LEAD-2 - Vertical &amp;amp; Multi-channels Peristaltic Pump</td><td>LONGER</td><td></td><td></td></tr><tr><td>Lightview XL&amp;nbsp;</td><td>Brightech</td><td></td><td></td></tr><tr><td>Methanol (Certified ACS)</td><td>Fischer Scientific</td><td>A412-4</td><td></td></tr><tr><td>Nikon D850</td><td>Nikon</td><td></td><td></td></tr><tr><td>NinjaTek NinjaFlex TPU @MK4</td><td>NinjaTek</td><td></td><td></td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch Laboratories</td><td>017-000-121</td><td></td></tr><tr><td>Original Prusa MK4 3D printer</td><td>Prusa Research</td><td></td><td></td></tr><tr><td>PAP pen</td><td>Abcam</td><td>ab2601</td><td></td></tr><tr><td>Paraformaldehyde, 32%</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td></td></tr><tr><td>Polycam</td><td>Polycam</td><td></td><td></td></tr><tr><td>Primary antibody</td><td></td><td></td><td></td></tr><tr><td>PrusaSlicer 2.7.1</td><td>Prusa Research</td><td></td><td></td></tr><tr><td>SARA-Engine</td><td>pita4 mobile LLC</td><td></td><td></td></tr><tr><td>Scaniverse</td><td>Niantic</td><td></td><td></td></tr><tr><td>Secondary antibody</td><td></td><td></td><td></td></tr><tr><td>SlowFade Gold Antiface Mountant</td><td>Invitrogen</td><td>S36936</td><td></td></tr><tr><td>Sodium azide, 5% (w/v)</td><td>Ricca Chemical Company</td><td>7144.8-32</td><td></td></tr><tr><td>SOMATOM Definition AS</td><td>Siemens Healthcare</td><td></td><td></td></tr><tr><td>Standard Field Surgi-Spec Telescopes,&amp;nbsp;</td><td>Designs for Vision</td><td></td><td></td></tr><tr><td>Stereomicroscope System SZ61</td><td>OLYMPUS</td><td></td><td></td></tr><tr><td>StereoPhoto Maker</td><td>Free ware developed by Masuji Suto</td><td></td><td></td></tr><tr><td>Superfrost Plus Microscope Slides, Precleaned</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-50ML</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416-100ML</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>534056-4L</td><td></td></tr><tr><td>Ziostation2</td><td>Ziosoft, AMIN</td><td></td><td></td></tr></tbody></table>

pipeline for multi-scale three-dimensional anatomic study of the human heart

对排斥移植的未衰竭人类心脏的详细研究为在微观和宏观尺度上进行结构分析提供了独特的机会。这些技术包括组织透明化（对溶剂透明化器官进行改良免疫标记的三维 （3D） 成像）和免疫组织化学染色。中镜检查程序包括立体解剖和显微计算机断层扫描 （CT）。肉眼检查程序包括大体解剖、摄影（包括立体和摄影测量）、CT 和物理或虚拟解剖或整个心脏的 3D 打印。在肉眼检查之前，可以进行压力灌注固定以保持心脏的 3D 结构和生理相关形态。将这些技术相结合来研究人类心脏是独特且至关重要的，对于在心脏 3D 结构的背景下理解不同解剖特征（如冠状动脉系统和心肌神经支配）之间的关系至关重要。该协议详细描述了方法，并包括代表性结果，以说明人类心脏解剖学研究的进展。

微量检查 应用组织透明化允许使用共聚焦显微镜对更大体积的组织进行 3D 成像。在心脏中，可以观察到包含心脏神经元的神经节和心肌神经支配的神经模式（图2）。 图 3 显示了神经和平滑肌细胞免疫染色的人左心室心肌的共聚焦图像。观察到血管穿过心肌，并鉴定出许多神经纤维，既与血管相关又独立于血管。
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作者聚焦:通过心脏多尺度分析推进人类心脏解剖学

该协议提供了一个全面的管道，用于分析从人类心脏获得的样品，这些样品跨越微观和宏观尺度。

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 ...

pipeline-for-multi-scale-three-dimensional-anatomic-study-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

339 次观看.  David Geffen School of Medicine at UCLA. 了解心脏解剖结构是了解心脏生理学的基础。功能解剖学的研究已经跨越了微观到宏观尺度，但主要使用动物心脏。对排斥移植的供体人类心脏的系统研究为开发涵盖微观到宏观尺度的综合图谱提供了独特的机会。通过对不同尺度的人类心脏的研究，我们展示了心脏的精细神经支配，确定了心脏解剖结构以改进手术和介入方法，并开发了解剖学...

视频: 作者聚焦:通过心脏多尺度分析推进人类心脏解剖学

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

科研

生物工程

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

发育生物学

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

神经科学

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D全心脏心肌组织分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

医学

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

axolotl 中的二维和三维超声心动图

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

用于全面心脏成像的光片显微镜和组织透明化

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

光片成像揭示啮齿动物心脏的心脏结构

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

用于人类心脏多尺度三维解剖学研究的管道

副本

摘要

探索更多视频

此视频中的章节

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

3D全心脏心肌组织分析

在微流体平台内开发 3D 组织人类心脏组织

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

axolotl 中的二维和三维超声心动图

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

用于人类心脏多尺度三维解剖学研究的管道

副本

摘要

探索更多视频

此视频中的章节

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

3D全心脏心肌组织分析

在微流体平台内开发 3D 组织人类心脏组织

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

axolotl 中的二维和三维超声心动图

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo