这项工作部分得到了生物医学工程和技术中心的支持:NIH （1U01HL116321） 和 （1R01HL142290） 和美国心脏协会 10SDG4030042 （GZ）， 19POST34450156 （HCJ）.

所有动物护理均符合马里兰大学巴尔的摩分校的指导方针和机构动物护理和使用委员会批准的协议。
1. 制定解决方案
注意:提前准备解决方案。实验中使用了两种基本溶液:（1） 用于沐浴超富的生理盐水溶液 （PSS）和 （2） 泰罗德用于流明香水的溶液。需要用 CO2 连续冒泡才能保持 PSS 的 pH。HEPES 缓冲的 Tyrode 溶液用于流明而不是 PSS，以避免气泡进入容器，因为气泡会损坏内皮细胞7 并阻止流动。
<ol><li>PSS 解决方案:请参阅表 1中 PSS 解决方案的公式和组成。制作 10 升 Ca2+无葡萄糖无葡萄糖 PSS 库存解决方案，并在室温下存放。1小时在任何程序之前，拿出1升的股票溶液和泡沫与5%的CO 2（74%N2和21%O2），以保持pH的解决方案在〜7.4。添加 1.8 葡萄糖和 1.8 mL CaCl2 （1M 库存）8. 注:仅在pH为+7.4时冒泡后添加Ca2+， 否则，Ca2+ 将被沉淀。</li>
	<li>泰罗德的解决方案:在表1中查看泰罗德解决方案的公式和组成。过滤溶液（0.22μm），并储存在4°C，供将来使用9。</li>
	<li>泰罗德的解决方案与BDM（2，3-丁尼迪翁单体）:添加BDM作为可逆肌溶酶抑制剂，以防止收缩的肌细胞10。重量 600 毫克 BDM，并添加到 200 mL 的泰罗德溶液。用 BDM 搅拌溶液，直到完全溶解。无需进一步过滤。</li>
	<li>将包含 BDM 的 Tyrode 解决方案存储在 4 °C 以供将来使用。</li>
</ol>2. 室内准备
<ol><li>按照制造商的说明，提前用多二甲基硅氧烷 （PDMS） 涂上解剖室和实验室。</li>
	<li>用包含 BDM 的 Tyrode 溶液填充解剖室。</li>
	<li>手术前1小时打开冷水机组。将温度设置为 4 °C。</li>
</ol>3. 坎努拉准备
<ol><li>打开微便士拉拔器。根据制造商的说明选择设置。</li>
	<li>拿一个干净的玻璃管（使用的玻璃管的外径和内径分别为1.2毫米和0.69毫米）。</li>
	<li>将玻璃管插入带有 U 形铂加热灯丝的 Sutter 移液器拉拔器的凹槽夹子中。</li>
	<li>激活拉拔器以产生两个管子，每个管子都有一个细长的尖端。</li>
	<li>在解剖显微镜下使用一把细细的剪刀切割每个管子的尖端，使尖端的最终直径在 100 到 150μm 之间。</li>
	<li>火抛光切管的尖端，稍微圆尖锐的边缘。</li>
	<li>使用铂线（用精细控制加热）沿着轴弯曲管，该线位于轴的侧面，距离尖端约 2 毫米。管子的弯曲角度应在 45° 左右。</li>
	<li>将刀具的开放端插入压力肌图室的支架，并拧紧配件。将装有 Tyrode 解决方案的 5 mL 注射器连接到配件的另一侧。将管子连接到安装在机舱上的微操纵器（见图 1和图2）。</li>
	<li>使用注射器 （5 mL） 将 Tyrode 的溶液填充罐中，冲洗它、气泡的管子和连接器。</li>
	<li>在制罐程序之前，测量管内的注水压力超过给定的流量范围（50 至 300 μL/min， 图 3）。只有在使用新手杖时才这样做。 注:管内的注水压力与流量成正比，并符合线性（图3）。</li>
</ol>4. 提取鼠标心脏
<ol><li>将一只C57BL/6小鼠（性别，8-16周大）放入麻醉盒中，麻醉盒与异丙苯丙和氧的罐体相连。</li>
	<li>打开氧气罐，启动异氟化气的流动。等待~5分钟，直到鼠标完全麻醉。</li>
	<li>麻醉后，将鼠标从麻醉盒中取出，将肝素IP（注射到腹腔，360单位，0.5 mL/鼠标），以避免血管内血块形成。然后把鼠标放回麻醉盒。</li>
	<li>注射肝素10分钟后，将鼠标移到加热床上，用贴标签胶带稳定其爪子。使用鼻锥用异氟兰将鼠标完全麻醉。</li>
	<li>用钳子将鼠标的腹部皮肤抬起隔膜上方，并用手术剪刀切割和暴露隔膜。</li>
	<li>切膜片和胸骨。打开胸膛</li>
	<li>解剖胸腔的心脏，尽可能靠近胸壁。</li>
	<li>将心脏放入含有30mBDM的冰冷泰罗德溶液中。</li>
	<li>清洁心脏以去除肺等结缔组织。</li>
	<li>将心脏转移到充满Tyrode含有30 mM BDM的溶液的预冷解剖室。</li>
</ol>5. 隔膜动脉的准备和凝固
<ol><li>打开伺服泵。将伺服泵设置为"流"模式。让它高速运行，直到管子充满Tyrode的溶液，将用于渗透血管流明。确保管子里没有气泡。然后降低速度。</li>
	<li>将心脏固定在 PDMS 上，避免对心脏中间区域造成任何损伤。</li>
	<li>删除右侧和左侧。切开右心室，使用解剖显微镜拆下右心室自由墙。</li>
	<li>暴露和识别隔膜动脉。将尼龙线（直径30μm）放置在隔膜动脉下，并系上松结，供将来使用。 注:使用解剖显微镜可以轻松识别隔膜动脉。在大多数情况下，隔膜动脉是来自右冠状动脉的隔膜上的主要动脉。</li>
	<li>使用细剪刀移除左心室自由墙。</li>
	<li>将肌制备转移到放置管子的实验室中。房间内装满了泰罗德含有 BDM 的溶液。这个房间的底部涂有一层薄薄的（约2毫米）的PDMS。</li>
	<li>调整管子的位置（使用微操纵器），使隔膜动脉的坎化。拧紧结。</li>
	<li>使用小针固定在室底的肌，使显微镜目标能够清楚地看到血管。注意管子的角度和位置，确保管子的尖端与动脉壁平行。</li>
	<li>通过通过管子逐渐从注射器中排出溶液来测试罐装。如果所有元素都正常工作，肌中的残余血液将在这个过程开始时退出组织，只有 Tyrode 的溶液稍后才能看到。</li>
</ol>6. 稳定准备
<ol><li>打开提供超级输液解决方案的离心泵。然后以 3-4 mL/min 的速度在浴缸中不断用预加气的 PSS 将制剂灌装。</li>
	<li>打开超级输液解决方案的温度控制器。将浴缸的温度调整为~35-37°C。</li>
	<li>将管子连接到伺服泵。阅读压力伺服控制器上显示的压力。</li>
	<li>监控流量和压力。流量 （μL/min） 和动脉注水压力 （mm Hg） 使用数字化器进行数字化，并使用相关软件进行记录（图 2）。</li>
	<li>调整流量以设置压力~10 mmHg，并让它运行约10分钟。</li>
	<li>增加发光溶液的流量，使动脉压力~60 mmHg。通过减去管子的压力下降（图3），获得动脉本身的最后灌注压力。 注:如果选择压力伺服控制器上的"压力"模式，这些实验的发光压力设置为~60 mmHg。然后监视流量的变化。</li>
	<li>通过监测流量和/或压力水平，让样品稳定约 30-60 分钟。动脉压力的逐渐增加通常观察到在罐装后注入的开始。一个新的稳定状态将在大约30分钟内自行建立（图4）。</li>
	<li>在注水压力稳定后（在恒定流量下）启动实验。</li>
</ol>7. 用荧光标记的小麦胚芽凝胶素 （WGA） 装载制剂
<ol><li>通过将 100μg 的连体 WGA 添加到泰罗德溶液的 5 mL 中，准备泰罗德包含 Alexa-Fluor-488 结合 WGA 的解决方案。</li>
	<li>小心地将香水从普通泰罗德的溶液切换到含有荧光标记的WGA的溶液中。重要的是要小心地切换香水，以便不引入气泡。</li>
	<li>在大约 30 分钟的 WGA 注入或 WGA 解决方案即将用完时，将香水变回 Tyrode 的正常溶液。</li>
</ol>8. 动脉和毛细血管的共生成像
<ol><li>打开尼普考磁盘心目显微镜系统。</li>
	<li>使用显微镜在传输光模式下使用低功耗目标 （4 倍） 定位隔膜动脉。 注:塞普塔尔动脉可以通过跟随管子的位置找到。</li>
	<li>开始与旋转盘共振成像，并选择488纳米激发激光器，40倍客观放大，并调整激光强度和采样率（每张图像10-500毫秒）。</li>
	<li>为共生显微镜设置顶部和底部成像位置，以定义成像范围并输入 z 堆栈成像的参数。</li>
	<li>根据正在使用的系统的建议设置步幅大小或根据需要设置步幅大小。</li>
	<li>选择 z 堆栈设置和/或时间系列设置。</li>
	<li>选择或设置一个新文件夹来存储新图像。</li>
	<li>单击"运行"以开始录制成像以供将来分析（图 5）。</li>
</ol>9. 示例血管除尘器实验:针刺引起的血管化（视频 1）。
<ol><li>准备皮纳西迪尔库存溶液（DMSO中的100 mM），并存储在4°C。</li>
	<li>准备 10 mL 的泰罗德解决方案。加入 10 μL 的皮纳西迪尔库存溶液，使皮纳西迪尔最终浓度达到 100 μM。</li>
	<li>想象低放大率的肌（ 4 倍目标），包括隔膜动脉和分支动脉。</li>
	<li>将发光溶液切换到含有小黄素的溶液中。</li>
</ol>10. 例血流量控制实验:血管收缩器引起的动脉输液压力在恒流时增加（图6）
<ol><li>准备内啡林-1 （ET-1） 库存溶液 （10 μM）， 并存储在 -20 °C。</li>
	<li>准备 10 mL 的泰罗德解决方案。添加 10 μL 的 ET-1 股票解决方案 （10 μM），使 ET-1 的最终浓度达到 10 nM。</li>
	<li>记录光压与恒定的流动和图像的肌肉。</li>
	<li>将发光溶液切换到包含解决方案的 ET-1。</li>
</ol>11. 毛细木带毛细的示例图像 （图 7）
<ol><li>牺牲本协议第 4 节中描述的 NG2DsRedBAC 转基因鼠标。</li>
	<li>提取心脏，封装隔膜动脉，并按照协议5-7加载样品与亚历克萨-弗鲁尔-488结合WGA。</li>
	<li>想象肌肉在 488 nm 和 560 nm 兴奋，和 510 - 525 nm ， 575 - 625 nm 排放使用腹膜 .动脉压力将在40 mmHg左右。</li>
</ol>

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在目前的工作中，我们引入了一种非常简单但非常实用的外生体方法，以研究生理条件下心脏的冠状微循环。这种方法是从机械调查修改使用大鼠2。具有挑战性的新增技术是高速和高光学分辨率的成像技术。因此，我们能够利用目前市售的先进光学成像系统。通过仔细解剖和放置功能肌肉准备在有利的位置，我们能够可视化动脉，毛细血管前动脉，毛细血管，以及腹膜，并能够...

适当的冠状动脉压力流量调节可保证心脏有足够的血液供应，以满足其代谢需求1.然而，尽管过去几十年在体内和体外进行了广泛的研究，但最近才清楚冠状动脉压力流在心脏中是如何动态调节的。原因之一是由于心脏的不断跳动，难以为此类研究建立生理工作模式。无论如何，已经建立了各种方法来观察活组织或动物的冠状微血管，但这些方法都未能达到恒定/稳定的焦点和测量压力，流量和微血管直径在同一时间2，3。10年前，在跳动的心脏中引入了冠状动脉微血管的直接可视化，但小血管的直径测量具有挑战性，许多与微循环相关的专门细胞类型的特定功能同样令人烦恼。即使是频闪法和浮动目标系统也不能同时提供上述信息。然而，使用上述技术已获得大量有价值的资料，这有助于我们更深入地了解冠状动脉血流的调节。本文描述的方法将帮助人们详细研究和理解冠状动脉、动脉和微血管的成分对刺激和代谢需求的反应。
我们为开展这些研究而建立的工作模式是建立在韦斯特霍夫等人先前的工作基础上的。在小鼠心脏的隔膜动脉凝固后，生理盐水溶液用于渗透动脉，以保持肌细胞和心脏组织的其他成分滋养。使用适当的荧光指示器监测动脉注水压力、流量和血管直径等生理功能。该方法使我们能够在活组织生理压力下可视化冠状微血管床，并首次研究微循环调节背后的细胞机制。

<table><tbody><tr><td>1 M CaCl2 solution</td><td>MilliporeSigma, USA</td><td>21115</td><td></td></tr><tr><td>1 M MgCl2 solution</td><td>MilliporeSigma, USA</td><td>M1028</td><td></td></tr><tr><td>AxoScope software</td><td>Molecular Devices, San Jose, CA, USA</td><td></td><td></td></tr><tr><td>Chiller/water incubator</td><td>FisherScientific, USA</td><td>Isotemp 3016S</td><td></td></tr><tr><td>Confocal</td><td>Nikon Instruments, USA</td><td>A1R</td><td></td></tr><tr><td>Custom glass tubing</td><td>Drummond Scientific Company</td><td>9-000-3301</td><td></td></tr><tr><td>Digidata 1322A</td><td>Molecular Devices, San Jose, CA, USA</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus, Japan</td><td>SZX12</td><td></td></tr><tr><td>Endothelin-1</td><td>MilliporeSigma, USA</td><td>E7764</td><td></td></tr><tr><td>Forceps</td><td>Fine Scientific Tools</td><td>11295-51</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Sigma-Aldrich, USA</td><td>H3393</td><td></td></tr><tr><td>Inline solution Heater</td><td>Warner Istruments, Hamden, CT, USA</td><td>SH-27B</td><td></td></tr><tr><td>Isoflurane</td><td>VETone, Idaho, USA</td><td>502017</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments, Novato, CA, USA</td><td>P-97</td><td></td></tr><tr><td>Micropipette/cannula holder</td><td>Warner Istruments, Hamden, CT, USA</td><td>64-0981</td><td></td></tr><tr><td>NG2DsRedBAC transgenic mouse</td><td>The Jackson Laboratory</td><td>#008241</td><td></td></tr><tr><td>Nylon thread for tying blood vessels</td><td>Living Systems Instrumentation, Burlington, Vt, USA</td><td>THR-G</td><td></td></tr><tr><td>PDMS (polydimethylsiloxane)</td><td>SYLGARD, Germantown, WI, USA</td><td>184 SIL ELAST KIT</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson, Middleton, WI, USA</td><td>minipuls 3</td><td></td></tr><tr><td>Pressure Servo Controller</td><td>Living Systems Instrumentation, Burlington, Vt, USA</td><td>PS-200-S</td><td></td></tr><tr><td>Scissors</td><td>Fine Scientific Tools, Foster City, CA, USA</td><td>15000-10</td><td></td></tr><tr><td>Servo Pump</td><td>Living Systems Instrumentation, Burlington, Vt, USA</td><td>PS-200-P</td><td></td></tr><tr><td>Temperature controller</td><td>Warner Instruments, Hamden, CT, USA</td><td>TC-324B</td><td></td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td><td>ThermoFisher Scientific, Waltham, MA USA</td><td>W11261</td><td></td></tr></tbody></table>

dynamic measurement and imaging of capillaries, arterioles, and pericytes in mouse heart

冠状动脉色调以及毛细血管的开启或关闭在很大程度上决定了在不断的输液压力向心肌细胞的血液。然而，很难监测整个心脏冠状动脉和毛细血管的动态变化，这主要是由于它的运动和不停的跳动。在这里，我们描述了一种方法，使监测动脉输液率，压力和直径变化的动脉和毛细血管在小鼠右心室肌肉。鼠标隔膜动脉在恒定的流量或压力下与另一个动态测量一起凝结和灌注。在用荧光标记的叶酸（例如， Alexa Fluor - 488 或 - 633 标记为小麦 - 格姆阿胶素， WGA ））进行注水后，右心室肌和隔膜中的动脉和毛细血管（和其他血管）很容易成像。然后，在存在或没有心脏收缩的情况下，可以测量容器直径的变化。当基因编码的荧光蛋白表达时，可以监测特定特征。例如，在表达NG2-DsRed的鼠标心脏中可视化了心率。该方法为研究毛细毛虫在心脏的生理功能提供了有益的平台。它还适用于通过同时测量血管/毛细管直径和动脉发光压力来研究试剂对心脏血流量的影响。这种准备，结合最先进的光学成像系统，使人们能够研究血液流动及其控制在细胞和分子水平的心脏在近生理条件下。

当荧光血管标记在血管流明中灌注时（这里 WGA 与 Alexa Fluor-488 结合），就可以使用高速共生显微镜可视化 图 5（ 左面板）中显示的整个血管树。进一步的放大使毛细的成像细节（图5，右面板）。由于加压系统支持对发光压力的持续监测，这种制剂可用于将动脉直径的变化与动脉压力关联使用。 视频 1 显示，当从流明中送达对 ATP 敏感的<s...

Watch this Scientific Journal Video about Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart at JoVE.com

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

这里介绍的是一个协议，通过前体监测维持压力的动脉灌注压力和流量，以及血管树成分，包括毛细管床和腹膜，研究活体murine心脏组织中的冠状微循环，因为隔膜动脉是可控和加压的。

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

dynamic-measurement-imaging-capillaries-arterioles-pericytes-mouse

Research

JoVE Journal

Medicine

5.3K 次观看.  University of Maryland School of Medicine. 这里介绍的是一个协议，通过前体监测维持压力的动脉灌注压力和流量，以及血管树成分，包括毛细管床和腹膜，研究活体murine心脏组织中的冠状微循环，因为隔膜动脉是可控和加压的。

视频: Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

医学

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

左心房，主动脉和肺动脉的超声心动图小鼠

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

通过心血管磁共振成像定量小鼠心脏左心室功能、心肌劳损和血流动力学力

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

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 ...

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

摘要

探索更多视频

在小鼠心脏的动作电位和钙瞬变的光学测绘

微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装

光片荧光显微术在小鼠心脏研究中的应用

左心房，主动脉和肺动脉的超声心动图小鼠

超声心动图与小鼠心脏形态学的组织学检查

通过心血管磁共振成像定量小鼠心脏左心室功能、心肌劳损和血流动力学力

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

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