Name: high-frequency ultrasound echocardiography to assess zebrafish cardiac function
Uploaded: 2020-03-12T15:00:00
Description: Watch this Scientific Journal Video about High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function at JoVE.com

我们感谢弗雷德·罗伯茨的技术支持和对手稿的修订。

涉及斑马鱼的所有程序均得到我们的机构动物护理和使用委员会的批准，并符合美国农业部动物福利法。
1. 实验设置
<ol><li>
设置图像采集平台
	<ol>
<li>使用小剪刀或手术刀在12点钟位置的海绵上切开，在扫描过程中握住鱼。将海绵放入玻璃容器中（图2A）。 注：切口的位置应留出足够的空间来移动传感器，并在平台倾斜进行...

<ol>
	<li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> Journal of Clinical Investigation. 122 (7), 2337-2343 (2012).</li><li>Verkerk, A. O., Remme, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+novel+research+tool+for+cardiac+(patho)electrophysiology+and+ion+channel+disorders.">Zebrafish: a novel research tool for cardiac (patho)electrophysiology and ion channel disorders.</a> Frontiers in Physiology. 3, 255 (2012).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovascular research. 91 (2), 279-288 (2011).</li><li>Poon, K. 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T., Pomerantsev, E. V., Mably, J. D., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+cardiovascular+function+confirms+functional+orthology+of+myocardial+contractility+pathways+in+zebrafish.">High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish.</a> Physiologycal Genomics. 42 (2), 300-309 (2010).</li><li>Mishra, S., 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=Human+amyloidogenic+light+chain+proteins+result+in+cardiac+dysfunction,+cell+death,+and+early+mortality+in+zebrafish.">Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.</a> American Journal of Physiology Heart Circulatory Physiology. 305 (1), H95-H103 (2013).</li><li>Ernens, I., Lumley, A. I., Devaux, Y., Wagner, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Coronary+Ultrasound+Imaging+to+Evaluate+Ventricular+Function+in+Adult+Zebrafish.">Use of Coronary Ultrasound Imaging to Evaluate Ventricular Function in Adult Zebrafish.</a> Zebrafish. 13 (6), 477-480 (2016).</li><li>Gonz&#225;lez-Rosa, J. M., 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=Use+of+Echocardiography+Reveals+Reestablishment+of+Ventricular+Pumping+Efficiency+and+Partial+Ventricular+Wall+Motion+Recovery+upon+Ventricular+Cryoinjury+in+the+Zebrafish.">Use of Echocardiography Reveals Reestablishment of Ventricular Pumping Efficiency and Partial Ventricular Wall Motion Recovery upon Ventricular Cryoinjury in the Zebrafish.</a> PLoS One. 9 (12), (2014).</li><li>Huang, C. C., Su, T. H., Shih, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+tissue+Doppler+imaging+of+the+zebrafish+heart+during+its+regeneration.">High-resolution tissue Doppler imaging of the zebrafish heart during its regeneration.</a> Zebrafish. 12 (1), 48-57 (2015).</li><li>Kang, B. J., 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=High-frequency+dual+mode+pulsed+wave+Doppler+imaging+for+monitoring+the+functional+regeneration+of+adult+zebrafish+hearts.">High-frequency dual mode pulsed wave Doppler imaging for monitoring the functional regeneration of adult zebrafish hearts.</a> Journal of the Royal Society Interface. 12 (103), (2015).</li><li>Lee, J., 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=Hemodynamics+and+ventricular+function+in+a+zebrafish+model+of+injury+and+repair.">Hemodynamics and ventricular function in a zebrafish model of injury and repair.</a> Zebrafish. 11 (5), 447-454 (2014).</li><li>Sun, L., Lien, C. L., Xu, X., Shung, K. 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描述了一种系统的方法，用于对成年斑马鱼进行心电成像和心脏功能评估。回声心动图是活成年鱼心脏成像和功能分析中唯一可用的非侵入性和最可靠的方法，在斑马鱼心血管研究中越来越受欢迎。所需时间很短，允许进行高通量和纵向研究。然而，所使用的方法和数据分析存在相当大的差异。当如此多的变量会影响即将到来的参数时，斑马鱼回声心动图的标准化是非常困难的。在进行实验研究?...

斑马鱼（Danio rerio）是研究发育过程和人类疾病的成熟脊椎动物模型1。斑马鱼在胚胎发育过程中具有与人类（70%）的遗传相似性（70%）、遗传可感染性、高繁殖性和光学透明度，允许对器官和组织（包括心脏）进行直接视觉分析。尽管只有一个中庭和一个心室，斑马鱼的心脏（图1）在生理上类似于哺乳动物的四室心脏。重要的是，斑马鱼的心率、心电图形态和动作潜能形状比人类比鼠种更像2。这些特点使斑马鱼成为心血管研究的优秀模型，为心脏发育33、4、4再生5和病理状况11、3、43,4提供了重要见解，包括动脉硬化、心肌病、心律失常、先天性心脏病和淀粉样蛋白光链心肌毒性1,1、4、6。,6通过高速视频显微镜77、88的直接视频分析，在胚胎阶段（受精后1天）可以评估心脏功能。然而，斑马鱼在胚胎阶段后失去透明度，限制了正常成熟心脏和晚发心脏状况的功能评估。为了克服这一限制，Echocardiix被成功地用作高分辨率、实时、非侵入性成像的替代品，以评估成年斑马鱼的心脏功能9,109、10、11、12、13、14、15。,11,12,13,14,15
在斑马鱼中，心脏位于胸腔中，紧接在刺的后部，中庭位于心室的背对背。中庭从窦静脉吸血中收集静脉血，并将其转移到心室，在那里进一步泵送至球状动脉（图1）。在这里，我们描述了一种生理、水下、协议，通过非侵入性超声心动图评估成年斑马鱼的心脏功能，使用中心频率为50 MHz的线性阵列超声探头，以30μm的分辨率进行B模式成像。由于超声波很容易在水中传播，因此在鱼和水下扫描探头之间保持接近，为心脏检测提供足够的接触面，无需超声波凝胶，而且对鱼的总体压力较小。虽然一些作者9、12、1312,13报告了替代斑马鱼超声心动系统，但在这里我们介绍了适用于动物高频超声的通用和最常用的设置。
该方法允许对成年斑马鱼心脏进行高分辨率成像，追踪心脏结构，并从多普勒血流测量中量化峰值速度。我们在体内显示重要的收缩和舒张参数的可靠定量，如弹出分数 （EF）、小数区域变化 （FAC）、心室血液流入和流出速度、心率 （HR） 和心脏输出 （CO）。我们帮助建立一个可靠的正常健康成年斑马鱼心脏功能和尺寸参数的范围，以便更精确地评估病理状态。总体而言，我们为评估斑马鱼的心脏功能提供了一种可靠的方法，在建立和验证斑马鱼心脏病模型66、16、16心脏损伤和恢复10、13,13和再生11、12,12中非常有用，并可用于进一步评估潜在的药物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Double sided tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fish net</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass container - 100 inch high</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High frequency transducer</td>
			<td>Fujifilm/VisualSonics</td>
			<td>MX700</td>
			<td>Band width 29-71 MHz, Centre transmit 50 MHz, Axial resolution 30 &#181;m</td>
		</tr>
		<tr>
			<td>Plastic teaspoon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel or scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small fish tanks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge (kitchen sponge)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets (graduated 3 mL)</td>
			<td>Samco Scientific</td>
			<td>212</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 3100 Imaging system and imaging station</td>
			<td>Fujifilm/VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo LAB sofware v 1.7.1</td>
			<td>Fujifilm/VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-frequency ultrasound echocardiography to assess zebrafish cardiac function

斑马鱼（Danio rerio）已成为心血管研究中非常受欢迎的模型有机体，包括人类心脏病，这主要是因为它的胚胎透明度、遗传性和快速、高通量研究的便利性。然而，透明度的丧失限制了成人阶段的心脏功能分析，这使得与年龄相关的心脏病的建模复杂化。为了克服这些限制，斑马鱼的高频超声超声心动图正在成为一个可行的选择。在这里，我们提出了一个详细的协议，通过非侵入性超声心动图，使用高频超声来评估成年斑马鱼的心脏功能。该方法允许对斑马鱼的心脏尺寸进行可视化和分析，并量化重要的功能参数，包括心率、中风体积、心脏输出和弹出分数。在这种方法中，鱼被麻醉并保存在水下，手术后可以回收。虽然高频超声是一种昂贵的技术，但相同的成像平台可以通过调整不同的传感器来用于不同的物种（例如，鼠类和斑马鱼）。斑马鱼回声心动图是心脏排便的有力方法，可用于疾病模型，特别是晚期疾病的验证和表征;毒品屏幕;和心脏损伤，恢复和再生能力的研究。

所述协议允许测量重要的心电图和功能参数，类似于人类和动物回声心动图中使用的技术。B-Mode 图像允许跟踪心室内壁（图 5），并获取尺寸数据（如室和壁尺寸）和功能数据（如心率、冲程体积和心脏输出）以及心室收缩函数的参数，如小数面积变化和弹出分数（表 1）。使用彩色多普勒模式图像在心室瓣的测量也提供心室流入和流出...

Watch this Scientific Journal Video about High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function at JoVE.com

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

我们描述了一种使用高频回声心动图评估成年斑马鱼心脏形态和功能的协议。该方法允许心脏可视化和随后定量功能参数，如心率 （HR）、心脏输出 （CO）、小数面积变化 （FAC）、弹出分数 （EF） 以及血液流入和流出速度。

高频超声超声心动图评估斑马鱼心脏功能

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

该协议允许非侵入性和高分辨率的表示和分析成年斑马鱼的心脏功能。此方法可用于验证成人心脏病模型和潜在的药物屏幕。该技术允许鱼的快速定位，以正确的成像提供准确和可重复的结果。它是无创的，在水下进行，允许鱼的快速恢复。要设置图像采集平台，请使用小剪刀或手术刀在 12 点钟位置对海绵进行切口。将海绵放在玻璃容器中。使用双面胶带，将装有海绵的玻璃容器贴在超声波平台上。确保玻璃容器位于平台的中心并牢固连接。使用平台支架左侧的旋钮向前倾斜平台约 30 度。在玻璃容器中填充大约200毫升的鱼系统水，每毫升三分之一甲烷硫化酯含有0.2毫克。将传感器插入工作火车站的微操纵器支架内，将传感器的槽口转向操作员。使阵列与地面平行，工作侧与舞台保持纵向关系。在两侧留下足够的空间，让现在连接的传感器导轨系统沿 x 轴和 y 轴移动。登录到控制软件并选择鼠标小血管。为研究中诱导的每个动物创建一个新的研究以及一个新的系列。按浏览器页面上屏幕左下角的新研究按钮。视图以 B 模式启动。使用鱼网将鱼转移到一个装有系统水的小水箱中，每毫升三分鱼含有0.2毫克。等到鱼完全麻醉，没有运动或没有反应触摸。轻轻地快速将鱼与海绵一起转移到玻璃容器中，将鱼的腹侧朝上。使用导轨系统上的手柄轻轻降低传感器，将其纵向放置，并关闭鱼的腹侧，传感器的凹口朝向操作员。从鱼中留下两到三毫米的间隙。使用所有三个轴中的微操纵器调整与传感器的平台，直到鱼心被可视化。本地化心脏后，立即选择或停留在 B 模式，通过放大来缩小字段，以便更近距离地查看心脏。如果需要，通过将动态范围设置为 45 到 50 分贝来增强图像的质量或对比度。转到更多控件选项中的 B 模式控件，然后将更改为模式预设。点击模式预设，在开始新系列成像之前，每次选择优化的图像采集设置。在长轴平面上拍摄尽可能多的图像。接下来，切换到彩色多普勒，用于血流检测和采集。使用触摸屏，将象限放置在电顶阀的顶部，并定位由红色信号区分的流入。尽可能减少象限区域以增加帧速率。激活脉冲波多普勒移动到样本心室血液流入速度。将样品体积门放置在发动机舱阀中心，以检测最大流量。红色信号变得更黄。使用手指调整屏幕上的脉冲波角度，使其与血液流入的方向对齐。按"开始"或"更新"开始采样流入心室的血液速度。通过将彩色多普勒象限放置在心室和球茎之间的交界处并确定流出速度，并本地化流量。这通过蓝色信号进行区分。将样品体积栅极位于心室球形结交汇点前，并调整角度校正线以匹配血流方向。调整在流速面板中提升的基线，以便完全检测和跟踪信号峰值。一旦图像采集完成，使用茶匙，将鱼转移到常规系统中，无三水加水，让鱼恢复。为了帮助恢复，使用转移移液器反复在刺上喷水，以促进水和氧气的加气。它通常需要30秒到2分钟才能恢复刺运动和游泳。打开图像分析软件。选择一个图像并单击图像处理图标。调整图像的亮度和对比度，以便清晰地可视化心室壁或血流模式。使用 B 模式图像，从心脏包测量上的准星长轴选项打开下拉列表。选择LV微量和跟踪心室内壁在systole和舒舒斯托，以获得心室面积在systole和舒张和舒张体积和结束收缩体积。本研究认为，B模式图像允许跟踪囊肿和隔膜内壁的内壁，并获取室和壁尺寸等尺寸数据。还获得了心率、中风体积、心脏输出等功能数据，以及心室收缩功能参数，包括小数面积变化和弹出分数。使用彩色多普勒模式图像进行心室进气阀水平的测量也提供了心室流入和流出血液速度。基于脉冲波多普勒图像，通过跟踪连续三个大音流峰产生心率值。最重要的是正确定位超声波平台和鱼，以进行准确的成像，并最大限度地减少鱼对三元鱼的接触。支持此程序的其他方法包括鱼心电图，用于快速检测心脏功能障碍，如心律失常或组织学方法，以评估心脏组织结构或细胞死亡。这些技术允许评估斑马鱼的心脏功能，并在斑马鱼心脏病模型中建立心脏表型。它还允许测试新药，研究心脏损伤和再生能力。三金MS-222是一种皮肤、眼睛和呼吸刺激物。在准备三金溶液时，防护设备应包括防尘面具、眼罩和手套。

Research

JoVE Journal

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

科研

发育生物学

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

采用触摸诱发反应和运动测定以评估在斑马鱼肌肉性能和功能

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

建立有限元模型，研究斑马鱼下颌生物力学

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

斑马鱼高血糖的急性和慢性模型：评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

高频超声在胎儿和胎盘发育分析中的影响

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...