Name: dissection and observation of honey bee dorsal vessel for studies of cardiac function
Uploaded: 2016-12-12T13:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

The authors thank Drs. Jeffrey Bloomquist and Daniel Swale for their technical comments and suggestions. This project was partially funded by the Department of Entomology and the College of Agriculture and Life Sciences at Virginia Tech.

1.收集和考试科目的准备<ol><li>收集从菌落蜜蜂的合适数量。 注意:需要的数量取决于不仅大小和实验的范围，但也研究者的技能。例如，如果有2个治疗组，每组10蜜蜂期望样本大小，一个合理的熟练研究者可以收集至少30蜜蜂占不成功解剖，并用20有用的视频得分结束。 </li><li>最小化该集合和解剖之间经过的时间量。 注意:虽然蜂可以在实验室中被安置为之前夹层天（ 即 ，保持在一解剖背脉管稳定心脏速率的可能性）已观察解剖的成功率相对降低到的时间量蜜蜂被安置从殖民地了。 <ol><li>而收容在实验室提供的水和食物的来源蜜蜂。对于EXAmple，在最低限度，提供对50％（重量/体积）的水的蔗糖溶液（这是足够小于6小时的持续时间）。更长的时间，提供访问蜜蜂蜂蜜。 </li><li>房子蜜蜂在大约32°C和60-80％的相对湿度温度在实验室过夜，以减轻压力和避免脱水。 </li></ol></li><li>此前解剖，麻醉蜜蜂简要方便搬运。 注意:此可降低夹层的成功率并降低吞吐量。 <ol><li>寒意蜜蜂，方法是将它们放在冰上或成只是足够长的时间，以减少要想在方便搬运移动冰箱。 </li><li>另外，简单地揭露蜜蜂的 CO 2，为了方便搬运。 注:长期暴露在寒冷可减少解剖的成功率。延伸或重复暴露于CO 2也能降低夹层的成功率。 </li></ol></li></ol> 2.解剖背腹墙注:蜜蜂应在解剖时是活着的。 <ol><li>使用镊子和/或显微剪刀，删除的腿和翅膀，以促进腹部解剖。保持一个小烧杯或装有蒸馏水附近设有夹层之间的冲洗工具的目的类似容器。 </li><li>同时抑制与钳蜜蜂，利用显微切割剪刀沿第一和第二tergites之间背侧腹壁横向切割（参见图1）。 </li></ol><img alt="图1" src="/files/ftp_upload/55029/55029fig1.jpg" /> 图1:蜜蜂腹部背视图。初始切口应在第一和第二tergites之间进行，由红色线表示。比例尺= 1毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig1large.jpg" target="_blank">请点击她的</a>E要查看此图的放大版本。 <ol start="3"><li>同时轻轻夹紧用钳子第二背片的后缘，沿着从最初的切口，以托管架的蜂的每一侧纵向切割（参见图2）。切割时避免刺穿胃肠道要小心。 </li></ol><img alt="图2" src="/files/ftp_upload/55029/55029fig2.jpg" /> 图2:蜜蜂腹部的横向视图。第二和第三切口应沿着腹部的任一侧进行，由红色线表示。比例尺= 1毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li>交换剪刀第二组细镊子，并利用它们来仔细背腹壁从休息分离ÒF中的腹部。轻轻取出托管架和保持附着于背腹壁胃肠道的任何部分。避免破裂肠道，作为内容可以外套腹壁和妨碍背脉管的可视化。 </li></ol><img alt="图3" src="/files/ftp_upload/55029/55029fig3.jpg" /> 图3:背船只观。一旦肠道和托管架被去除，背容器是沿解剖背腹壁的中线可见。比例尺= 1毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>安排在照相机下方的所需取向背侧腹壁使得背脉管可见（ 见图3）。利用显微切割剪刀修剪掉多余的ABdominal墙阻碍背船只的可视化。背腹壁的形状应该像一个浅杯或碗当正确位于。 <ol><li>由于背脉管不延伸到蜂的最后面腹段，取出最后背片，以提高背脉管的可视化。 </li></ol></li><li>利用一个音量可调微量移液器，覆盖10微升等渗溶液，以保持生理条件和促进稳定的心跳背船只。 注:建议的解决方案是四分之一强度的林格氏溶液（0.120克氯化物/ L的钙，0.105mmol克/升氯化钾0.050克/升碳酸氢钠和2.250克/升氯化钠），已经发现，以促进稳定，连续心跳。 </li></ol> 3.观察和心率的调节<ol><li>允许背船只坐不受干扰，直到稳定，连续的心跳达到（通常在300秒）。 注:心跳可视化为背船只的节律性收缩。最初，可能似乎是没有心跳，特别是如果蜂麻醉，但心脏通常在等渗溶液休息几分钟后恢复跳动，并且可以继续搅打数小时，只要它仍然沐浴在溶液中。 </li><li>测量在每分钟（BPM）的节拍的数量方面的心脏速率。 <ol><li>记录在一个60秒的时间观察宫缩的数量。用一只手式计数器和一个计时器，以促进这一进程。 </li></ol></li><li>由前和用cardiomodulatory化合物处理之后记录所观察到的BPM测量心脏速率的变化。 注意:虽然所需要的时间来观察心脏速率的效果可根据化合物被测试而变化，在心脏速率的变化通常可在几分钟之内观察到。 <ol><li>确定基线心脏速率之前立即在加入任何试验化合物。 注:后处理心脏速率通常可以为90〜120秒后确定。 </li><li>通过在用于洗澡背容器相同等渗溶液中的化合物溶解制备潜在cardiomodulators（ 例如，章鱼胺）。 </li><li>试验化合物添加到通过利用micropipetter背脉管周围的溶液中。 </li></ol></li><li>获得更高的精度和更高的吞吐量，使每个测试受试者的录像，然后使用视频在稍后的时间得分心脏速率。 注意:这允许单个研究者错开解剖，以便于几乎连续生产的影片。当录制视频，建议最小长度为60秒大关添加任何测试化合物约240秒。这确保了研究者对得分基线心脏速率，然后对得分后处理心脏速率120秒另外60秒窗口60秒窗口治疗后。 </li></ol>

<ol>
	<li>Klowden, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+Systems.">Circulatory Systems.</a> Physiological Systems in Insects, 3rd Edition. , 365-413 (2013).</li><li>League, G. P., Onuh, O. C., Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structural+and+functional+analysis+of+the+larval+and+adult+dorsal+vessel+and+its+role+in+hemolymph+circulation+in+the+mosquito+Anopheles+gambiae.">Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae.</a> J Exp Biol. 218 (Pt 3), 370-380 (2015).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+Hemolymph+Circulation+in+the+Butterfly+Papilio-Machaon+L+Revealed+by+Contact+Thermography+and+Photocell+Measurements.">Oscillating Hemolymph Circulation in the Butterfly Papilio-Machaon L Revealed by Contact Thermography and Photocell Measurements.</a> J Comp Physiol. 139 (2), 145-163 (1980).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+circulation+and+tracheal+ventilation+in+holometabolous+insects.">Interaction of circulation and tracheal ventilation in holometabolous insects.</a> Adv Insect Physiol. 26, 297-351 (1996).</li><li>Schwab, E. R., Chilson, R. A., Eddleman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heartbeat+Rate+Modulation+Mediated+by+the+Ventral+Nerve+Cord+in+the+Honey-Bee,+Apis-Mellifera.">Heartbeat Rate Modulation Mediated by the Ventral Nerve Cord in the Honey-Bee, Apis-Mellifera.</a> J Comp Physiol B-Biochem Syst Environ Physiol. 161 (6), 602-610 (1991).</li><li>Kaiser, W., Weber, T., Otto, D., Miroschnikow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+supply+of+the+heart+and+electrocardiogram+potentials+with+reversed+polarity+in+sleeping+and+resting+honey+bees.">Oxygen supply of the heart and electrocardiogram potentials with reversed polarity in sleeping and resting honey bees.</a> Apidologie. 45 (1), 73-87 (2014).</li><li>Papaefthimiou, C., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine--a+single+modulator+with+double+action+on+the+heart+of+two+insect+species+(Apis+mellifera+macedonica+and+Bactrocera+oleae):+Acceleration+vs.+inhibition.">Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.</a> J Insect Physiol. 57 (2), 316-325 (2011).</li><li>Castro, C. A., Hogan, J. B., Benson, K. A., Shehata, C. W., Landauer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral-Effects+of+Vehicles+-+Dmso,+Ethanol,+Tween-20,+Tween-80,+and+Emulphor-620.">Behavioral-Effects of Vehicles - Dmso, Ethanol, Tween-20, Tween-80, and Emulphor-620.</a> Pharmacol Biochem Behav. 50 (4), 521-526 (1995).</li><li>Papaefthimiou, C., Papachristoforou, A., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+responses+of+the+honeybee+heart+to+nanomolar+concentrations+of+amitraz.">Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz.</a> Pestic Biochem Phys. 107 (1), 132-137 (2013).</li><li>Roeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+in+invertebrates.">Octopamine in invertebrates.</a> Prog Neurobiol. 59 (5), 533-561 (1999).</li><li>Johnson, E., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Drosophila+heartbeat+by+neurotransmitters.">Modulation of Drosophila heartbeat by neurotransmitters.</a> J Comp Physiol B. 167 (2), 89-97 (1997).</li></ol>

The authors have nothing to disclose.

The protocol presented here provides a simple and effective approach to testing pharmacological compounds for their effects on honey bee heart rate. As observed in prior experiments that either transect the ventral nerve cord of a living insect5 or dissect out the ventral nerve cord when exposing the dorsal vessel7, the loss of central nervous system regulation results in a stable, low frequency heartbeat. The low frequency of beats allows the investigator to visually assess heart rate without havin...

这种方法的总体目标是允许研究者快速，轻松地观察和量化药理剂对蜜蜂的心脏速率的影响。蜜蜂，像其他昆虫，具有传播血淋巴，血液的昆虫当量，在整个体腔，被称为血腔的开放循环系统。血淋巴的循环为营养物质，免疫因子，废产物，以及神经激素和其他信号分子1的运输是必不可少的。循环是通过背容器中，沿昆虫的背中线，以及附件搏动器官延伸促进。背容器被分成两个功能上不同的部分，指定在腹部的心脏，并在胸部和头部的主动脉。在对胸部和头部的心脏泵血淋巴传播收缩，而附属器官搏动血淋巴确保流向四肢。 <p类="jove_content"&gt;可以使用多种方法，这取决于昆虫的大小，生理学，或生命阶段观察到昆虫的心脏功能。观察幼虫或更小的昆虫心脏速率的常见方法是利用活体成像2。这种方法是在成年蜂不太有用，但是，由于它可能很难通过腹壁来清楚地观察背脉管。在各种昆虫，包括蜜蜂记录心脏速率的建立的方法，是利用接触热成像，其利用施加到昆虫的外部，以检测心脏脉动3,4-热敏电阻。在成人蜜蜂心脏速率也已使用电生理技术来测量电阻抗信号4,5-记录。这种技术需要的电极插入到动物旁边的心脏和使用阻抗变换器来记录心跳4。同样，心电图已用于DETECŧ由心脏产生，并与蜜蜂的录像相结合，观察心脏活动6变化的电信号。一个显着的优势，这些方法是心脏速率在一个完整的，活蜂评估，而不是在一个解剖的标本，这有助于确保全方位的主题生理反应的可用性。这些方法的挑战包括会计主体，需要限制之外的变量和刺激，可能改变心脏率，以及测试药物制剂时，确定合适的给药方法的固定或麻醉。 已用于研究蜂的心脏活动的另一种方法是，以部分解剖昆虫以暴露心脏，然后进行测量使用一个力位移传感器7背脉管收缩。在这个协议中，心脏不断与正在运行的生理盐水和测试沐浴合作mpounds可以溶解在该溶液应用到对象7。这种方法与前面描述的那些之间的显著区别在于腹神经索被去除，消除了对中枢神经系统已被证明在调节心脏速率5发挥作用。其结果是，在基线心跳，这通常是相当不稳定的，稳定在一个低得多的频率和幅度比在活昆虫5,7-通常观察到。什么所有这些方法的共同点在于，它们需要高度专业化和通常是昂贵的设备，除了专门知识的一定水平，以进行。或许最大的缺点是，没有这些方法均特别适用于涉及测试大量的受试者，如筛选潜在cardiomodulatory化合物文库的实验。 这里所描述的方法的最大优势是它的简单性。该协议是比较容易掌握，安装需要空间不大，只有最小的资金投入是必要的。该方法需要比一些蜜蜂，几个手术器械，等渗溶液更小，以及一个数字显微镜或用数码相机拍摄传统显微镜。蜜蜂解剖可视化背船只和数字视频使用前，用药剂治疗后，记录心脏速率。虽然录像实际上不是必要观察心脏速率的变化，这将大大增加吞吐量（ 即 ，可以在给定的时间量来处理对象的数目）。研究者可以一次录制了大量的视频，然后后来在更方便的时间攻入这些视频最大限度地提高效率。这种方法的另一个优点是，视频允许研究者重新开始，应在评分过程被中断，并使其更容易在Viewer，以便减少偏见蒙蔽治疗。

<table><tbody><tr><td>Dino-Lite Edge digital USB microscope</td><td>Dino-Lite</td><td>AM4815ZT</td><td>Any digital microscope or similar setup will suffice</td></tr><tr><td>Microscope stand</td><td>Dino-Lite</td><td>RK-10</td><td>Any stand appropriate for the digital microscope</td></tr><tr><td>Laptop or PC</td><td></td><td></td><td>Necessary for digital microscope</td></tr><tr><td>Microdissection scissors (Vannas, 8 cm, Straight, 5 mm Blades)</td><td>World Precision Instruments</td><td>14003</td><td>Any similar scissors suitable for microdissection will suffice</td></tr><tr><td>Microdissecting Forceps, 10.2 cm, Angled (2 pair)</td><td>World Precision Instruments</td><td>504482</td><td>Any similar forceps suitable for microdissection will suffice</td></tr><tr><td>Ringers solution 1/4 strength tablets&amp;nbsp;</td><td>Sigma-Aldrich</td><td>96724-100TAB</td><td></td></tr><tr><td>Dissecting tray</td><td></td><td></td><td>Any surface suitable for microdissection</td></tr><tr><td>Single channel 10 &amp;micro;l pipette</td><td></td><td></td><td>Any device capable of accurately delivering 10 &amp;micro;l volume</td></tr><tr><td>Pipette tips</td><td></td><td></td><td></td></tr><tr><td>Small beaker or container of water</td><td></td><td></td><td>Used to rinse instruments between subjects</td></tr><tr><td>Hand tally counter</td><td>Office Depot</td><td>295033</td><td>Any similar product will suffice</td></tr><tr><td>Timer</td><td>Office Depot</td><td>644219</td><td>Any similar product will suffice</td></tr><tr><td>Deionized water</td><td></td><td></td><td>Preparation of Ringers solution and rinsing instruments</td></tr></tbody></table>

dissection and observation of honey bee dorsal vessel for studies of cardiac function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

由于许多可能使用此协议待测试的药理活性化合物是不溶于水，必须有一个可靠的溶剂，将允许测试化合物通过用沐浴背脉管的等渗溶液被递送。二甲基亚砜（DMSO）是通常用作在动物8递送实验性药物和其他化合物的车辆的溶剂，并已成功地用于此目的的研究中检查农药对蜜蜂心脏活动9的效果。因此，DMSO被选为用于递送试验化合物于背容器，这需要它首?...

Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

解剖和蜜蜂背血管的观察心功能的研究

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

dissection-observation-honey-bee-dorsal-vessel-for-studies-cardiac

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.8K 次观看.  Virginia Tech. The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

视频: Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

科研

发育生物学

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

细胞周期变化的可视化和核测定心肌出生后

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

生物学

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and&#160;achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

The Drosophila heart sectioning and fluorescence imaging protocol simplifies studying heart structure and pathologies. This approach involves straightforward sectioning, staining, and imaging, bypassing the technical expertise needed for traditional dissection. It enhances accessibility, making Drosophila a more widely usable model for cardiac-related research within the broader scientific community.

使用冷冻切片进行 果蝇 心脏荧光染色的新型高效方法

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila ...

遗传学

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

心血管疾病大型动物模型中心脏的解剖技术和组织学取样

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

医学

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Heart Dissection: A Method to Observe Zebrafish Cardiac Development

Source: Singleman C. et. al., <a href="https://jove.unicartagenaproxy.elogim.com/v/3165/" target="_blank">Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio</a>. J. Vis. Exp. (2011).
This video describes a technique of isolating the heart from a zebrafish at multiple developmental stages to study cardiac development, maturation, and ageing.

心脏解剖:一种观察斑马鱼心脏发育的方法

Source: Singleman C. et. al., Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio. J. Vis. Exp. (2011).
This video describes a technique ...

解剖和蜜蜂背血管的观察心功能的研究

摘要

探索更多视频

此视频中的章节

在清除胚胎心脏冠状血管分析

细胞周期变化的可视化和核测定心肌出生后

可视化心脏跳动果蝇

在幼虫的监测心脏功能果蝇生理研究

使用冷冻切片进行果蝇心脏荧光染色的新型高效方法

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼

灭鼠工作心脏模型心肌的性能和氧耗的研究

心血管疾病大型动物模型中心脏的解剖技术和组织学取样

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

心脏解剖:一种观察斑马鱼心脏发育的方法

解剖和蜜蜂背血管的观察心功能的研究

摘要

探索更多视频

此视频中的章节

在清除胚胎心脏冠状血管分析

细胞周期变化的可视化和核测定心肌出生后

可视化心脏跳动果蝇

在幼虫的监测心脏功能果蝇生理研究

使用冷冻切片进行 果蝇 心脏荧光染色的新型高效方法

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼

灭鼠工作心脏模型心肌的性能和氧耗的研究

心血管疾病大型动物模型中心脏的解剖技术和组织学取样

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

心脏解剖:一种观察斑马鱼心脏发育的方法

使用冷冻切片进行果蝇心脏荧光染色的新型高效方法