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.

被験者の1収集と準備<ol><li>コロニーから蜂の適切な数を収集します。 注：必要な数は、規模や範囲の実験だけでなく、研究者のスキルだけでなく、依存します。 2つの治療グループでは、グループごとに10蜂の所望のサンプルサイズである場合たとえば、合理的に熟練した研究者は失敗した解剖を考慮して、少なくとも30蜂を集めるかもしれないし、スコアに20便利な動画で終わります。 </li><li>コレクションと解剖の間を通過する時間を最小にしてください。 注：ミツバチが解剖の前日間実験室に収容することができるが、解剖の成功率（ すなわち 、解剖背容器安定心拍数を維持する可能性）は、その時間の量に対して減少することが観察されていますミツバチコロニーから離れて収容されています。 <ol><li>研究室に収容されながら、水や食料の供給源とミツバチを提供します。エクサのためmple、少なくとも、（これ未満6時間の持続時間のために十分である）を水中でスクロースの50％（w / v）の溶液へのアクセスを提供します。より長い期間のために、ミツバチが蜜へのアクセスを提供します。 </li><li>ストレスを軽減し、脱水症状を避けるために、約32°C 60〜80％の相対湿度の温度で一晩ラボでハウス蜂。 </li></ol></li><li>解剖の前に、取り扱いを支援するために簡単にハチを麻酔。 注：これは、解剖の成功率を減少させ、スループットを減少させることができます。 <ol><li>ちょうど十分な長さの取り扱いを助けるために運動を減らすために、氷上や冷蔵庫にそれらを配置することにより、いずれかのミツバチを冷やします。 </li><li>また、簡単に取り扱いを助けるためにCO 2にミツバチを公開します。 注：寒さに拡張暴露は、解剖の成功率を減らすことができます。拡張またはCO 2への暴露を繰り返しても、解剖の成功率を減少させることができます。 </li></ol></li></ol> 2.解剖背側腹壁の注：蜂は解剖の時に生きている必要があります。 <ol><li>鉗子および/または顕微解剖ハサミを使用して、腹部の切開を容易にするための脚と翼を削除します。解剖の間に器具を洗浄する目的で、近くに蒸留水で満たされた小さなビーカーまたは類似の容器を保管してください。 </li><li>鉗子で蜂を抑制しつつ、第一および第二のtergites（ 図1参照）との間に背側腹壁に沿って横方向にカットする顕微解剖ハサミを使用しています。 </li></ol><img alt="図1" src="/files/ftp_upload/55029/55029fig1.jpg" /> 図1：ミツバチの腹部の背面図。赤線で示すように最初の切開は、第一および第二のtergitesとの間でなされるべきです。スケールバー= 1ミリメートル。 <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig1large.jpg" target="_blank">彼女をクリックしてください</a>eは、この図の拡大版を表示します。 <ol start="3"><li>軽くピンセットで第二背板の後縁を把持しながら、スティンガーに最初の切開から蜂の各辺に沿って縦方向に切断（ 図2を参照）。消化管を穿刺避けるために切断する際には注意してください。 </li></ol><img alt="図2" src="/files/ftp_upload/55029/55029fig2.jpg" /> 図2：ミツバチの腹部の側面図。赤線で示すように第二と第三の切開は、腹部の両側に沿ってなされるべきです。スケールバー= 1ミリメートル。 <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol start="4"><li>細かい鉗子の第二のセットのためのはさみを交換し、慎重に残りoから背側腹壁を分離するためにそれらを利用します腹部F。ゆっくりスティンガーと背側腹壁に付着したまま消化管の任意の部分を削除します。内容は背血管の可視化を妨げるコート腹壁をすることができているように、腸を破裂避けてください。 </li></ol><img alt="図3" src="/files/ftp_upload/55029/55029fig3.jpg" /> 図3：背容器の眺め。腸とスティンガーが削除されたら、背血管を切開背側腹壁の正中線に沿って表示されます。スケールバー= 1ミリメートル。 <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol start="5"><li>背容器が表示されるようにカメラの下に所望の向きで背側腹壁を配置します （ 図3を参照）。余分なABを離れてトリミングする顕微解剖はさみを利用背血管の可視化を妨げるdominal壁。背側腹壁の形状が適切に位置し、浅いカップまたはボウルのようになります。 <ol><li>背容器は蜂の最後尾腹部のセグメントに延びていないので、背の血管の可視化を向上させるために、最終的な背板を取り外します。 </li></ol></li><li>調整可能なボリュームのマイクロピペットを利用して、生理的条件を維持し、安定したハートビートを容易にするために、等張溶液10μlと背容器をカバーしています。 注：推奨されるソリューションは四半期強度リンゲル液（0.120グラム/ Lの塩化カルシウム、0.105グラム/ Lの塩化カリウム0.050グラム/ L重炭酸ナトリウム、および2.250グラム/ Lの塩化ナトリウム）安定を促進することが見出されています、連続ハートビート。 </li></ol> 3.観測と心拍数の調節<ol><li>安定した、連続的なハートビートが達成されるまで背容器を静置することを許可します（通常300秒以内）。 注：ハートビートは背容器のリズミカルな収縮として可視化されます。最初に、蜂が麻酔された場合は特に、ハートビートがないように見えるかもしれないが、心臓は通常、数分間等張液で休ん後に打撃を再開しますし、時間の暴行続けることができる、それが溶液に浸したまま提供。 </li><li>分あたりの拍数（BPM）の観点で心拍数を測定します。 <ol><li> 60秒の期間中に観察された収縮の回数を記録します。このプロセスを容易にするために、手の数取器とタイマーを使用してください。 </li></ol></li><li> cardiomodulatory化合物で処理する前と後に観察BPMを記録することによって、心拍数の変化を測定します。 注：心拍数への影響を観察するために必要な時間は、試験される化合物に依存して変化し得るが、心拍数の変化は、典型的には数分以内に観察することができます。 <ol><li>直前のベースライン心拍数を決定しますいずれの試験化合物を添加。 注：治療後の心拍数は、通常90〜120秒後に決定することができます。 </li><li>背の容器を浸して使用したのと同じ等張液中の化合物を溶解することによって潜在的なcardiomodulators（ 例えば、オクトパミン）を準備します。 </li><li>マイクロピペッターを利用して背容器を周囲の溶液に試験化合物を追加します。 </li></ol></li><li>より高い精度とより高いスループットのために、各被験者のビデオ録画を行い、その後、後で心拍数を獲得するためにビデオを使用しています。 注：これはビデオのほぼ連続生産を容易にするために解剖をずらすために、単一の調査員を可能にします。動画を記録する場合、最小推奨長さは、任意の試験化合物を60秒マークで追加されると、約240秒です。これは、研究者は、治療後の心拍数120秒をスコアのベースライン心拍数とし、別の60秒のウィンドウを採点するための60秒のウィンドウを持っていることを保証します治療後。 </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...

この方法論の全体的な目標は、研究者が迅速かつ容易に観察し、薬理学的薬剤は、ミツバチの心拍数に与える影響を定量化できるようにすることです。ミツバチは、他の昆虫のように、血体腔として知られている体腔、全体を通して、血リンパ、血液の昆虫同等のものを広めるオープン循環系を持っています。血リンパの循環が栄養素、免疫因子、老廃物、ならびに神経ホルモンおよび他のシグナル伝達分子1の輸送のために不可欠です。循環は昆虫の背側正中線だけでなく、アクセサリー拍動器官に沿って延びている背の容器、によって促進されます。背容器は腹部と胸部と頭の中で大動脈に心を指定、機能的に異なる2つのセクションに分かれています。胸部や頭部に向かって心臓ポンプ血リンパ中の伝播収縮は、付属拍動器官は、四肢への血リンパの流れを確保しながら。 <pクラス= &quot;jove_content&quot;&gt;昆虫心臓機能、サイズ、生理学、または昆虫のライフステージに応じて、様々な方法を使用して観察することができます。幼虫以下昆虫に心拍数を観察するための一般的な方法は、生体内イメージング2の使用です。それは、腹壁を通して背容器を明確に表示することは困難であるため、この方法は、しかしながら、成人ミツバチにおいてあまり有用です。ミツバチなどの昆虫、様々な心拍数を記録するための確立されたアプローチは、サーミスタは、心臓拍動3,4を検出するために、昆虫の外側に適用される利用接触サーモグラフィの使用です。成体ミツバチにおける心拍数は、電気インピーダンス信号4,5を測定するために電気技術を使用して記録されています。この技法は、心臓への次の動物に電極を挿入し、心拍4を記録するためのインピーダンス変換器の使用を必要とします。同様に、心電図はdetecするために使用されています心臓によって生成され、心臓の活動6の変化を観察するために蜂のビデオ録画と組み合わせた電気信号をtは。これらのアプローチの明確な利点は、心拍数ではなく、対象における生理学的応答の全範囲の可用性を保証するのに役立つ解剖標本、よりも、そのまま、生きている蜂で評価されていることです。これらのアプローチの課題は、被写体、心拍数を変化させるだけでなく、薬理学的薬剤をテストするときに、適切な配信方法を決定するかもしれない外部の変数と刺激を制限する必要の固定化や麻酔のための会計が含まれています。 ハチ心臓活動を研究するために使用されている別のアプローチは、部分的に、力変位変換器7を使用して、背部の血管収縮を測定し、その後、心臓を露出させるために、昆虫を分析することです。このプロトコルでは、心臓は継続的に生理食塩水やテストの共同の実行に浴びますmpoundsは、対象7への適用のために、この溶液中に溶解することができます。この方法及びそれらを前述の有意差は、腹側神経索は、中枢神経系は、心拍数5の調節に再生するために示されている役割を排除する、除去することです。結果は通常、非常に不安定なあるベースラインハートビートは、典型的には、生きている昆虫5,7で観察されるよりもはるかに低い周波数と振幅で安定するということです。どのようなこれらの方法の全てに共通しているの彼らが行われるために、ある程度の専門知識に加えて、高度に専門化し、しばしば高価な機器を必要とするということです。おそらく、最大の欠点は、これらのアプローチのいずれも、そのような潜在的にcardiomodulatory化合物のライブラリーをスクリーニングするように被験者の数が多い、試験伴う実験に特に適していないことです。 ここで説明されたアプローチの最大の強みそのシンプルさです。プロトコルは習得が比較的容易であり、セットアップは少しスペースを必要とし、最小限の金融入力が必要です。方法はいくつかの蜂、いくつかの手術器具、等張液、およびデジタル顕微鏡やデジタルカメラとの伝統的な顕微鏡のいずれかより少しが必要です。ハチは、背側血管を可視化するために解剖され、デジタルビデオは薬剤による治療前後の心拍数を記録するために使用されます。録画は、心拍数の変化を観察するために実際に必要ではないが、それは非常にスループットを増加させる（ すなわち 、一定時間内に処理可能な被験者の数）。研究者は一度にビデオの多数を記録し、後で都合のよいときにこれらのビデオを採点することによって、効率を最大限に高めることができます。このアプローチのもう一つの利点は、ビデオは調査員が採点処理が中断されるべきで、最初からやり直す、とvのためのそれを容易にすることを可能にするということですiewerバイアスを減少させるために治療を知らされます。

<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.

このプロトコルを使用して試験される可能性があり薬理学的に活性な化合物の多くは水溶性ではないので、試験化合物が背容器を浸すために使用される等張液を介して配信されることを可能にする信頼性の高い溶媒が必要です。ジメチルスルホキシド（DMSO）は、一般に、動物8に実験薬物および他の化合物を送達するためのビヒクルとして使用される溶剤であ...

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.

心機能の研究のための解剖と観察ハニービー背側の船

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 ビュー.  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.

ビデオ: 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.

クリア胚ハーツにおける冠状血管の分析

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

発生生物学

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.

生後心筋細胞における細胞周期変化の可視化と核の決意

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.

の心臓の鼓動の可視化ショウジョウバエ</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.

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

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.

クライオ切片による ショウジョウバエ の心臓蛍光染色のための新規で効率的な方法

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

遺伝学

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.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</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.

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

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.

循環器疾患大型動物モデルにおける心臓の解剖技術と組織学的サンプリング

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.

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（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.

心臓解剖:ゼブラフィッシュの心臓発達を観察する方法

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

心機能の研究のための解剖と観察ハニービー背側の船

要約

さらに動画を探す

この動画の章

クリア胚ハーツにおける冠状血管の分析

生後心筋細胞における細胞周期変化の可視化と核の決意

の心臓の鼓動の可視化ショウジョウバエ

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

クライオ切片によるショウジョウバエの心臓蛍光染色のための新規で効率的な方法

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

循環器疾患大型動物モデルにおける心臓の解剖技術と組織学的サンプリング

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（OCM）

心臓解剖:ゼブラフィッシュの心臓発達を観察する方法

心機能の研究のための解剖と観察ハニービー背側の船

要約

さらに動画を探す

この動画の章

クリア胚ハーツにおける冠状血管の分析

生後心筋細胞における細胞周期変化の可視化と核の決意

の心臓の鼓動の可視化ショウジョウバエ

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

クライオ切片による ショウジョウバエ の心臓蛍光染色のための新規で効率的な方法

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

循環器疾患大型動物モデルにおける心臓の解剖技術と組織学的サンプリング

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（OCM）

心臓解剖:ゼブラフィッシュの心臓発達を観察する方法

クライオ切片によるショウジョウバエの心臓蛍光染色のための新規で効率的な方法