Name: mechanical vessel injury in zebrafish embryos
Uploaded: 2015-02-17T14:00:00.000+00:00
Duration: 6 min 38 s
Description: Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

The authors would like to thank Drs. Stephen Wilson and Lisa Wilsbacher for helpful discussions. This work was supported in part by NIH HL054737.

注：ゼブラフィッシュを使用した手順は、UCSFの施設内動物管理使用委員会によって承認された。 ツールの調製<ol><li>ピンホルダーに特徴点のピンを挿入し、ピンをクランプ。 </li><li>先端の細い鉗子を使用して、慎重にわずかにフックを作成するために、ピンの先端を曲げる。 </li><li>負傷中の胚の操作および安定化のために、ラジオペンチを使用して、インスリン注射器に取り付けられた28ゲージ1/2インチの針の端を曲げ。 </li></ol>傷害のためのゼブラフィッシュ胚の作製<ol><li>ゼブラフィッシュの繁殖ペアを設定し、以前に22示すように、卵の水（60ug / mLの水族館塩）で卵を集める。 </li><li>胚はメラニン化を防ぐために、約8時間後に受精（HPF）であるとき、卵の水に0.003％、N-フェニルチオ尿素（PTU）を追加します。 </li><li>先端の細い鉗子を用いた実験に先立ってDechorionate 2日後の受精（DPF）の胚。 <br/&gt;注：循環開始後胚はいつでも怪我をすることができます。ここに示されたデータは、胚のdpf 2のためであるが、技術は成功し5のdpfまでの胚で適用されている。 </li><li> 0.02％緩衝化3-アミノ安息香酸（トリカイン）前の操作に約10分を有する胚を麻酔。 </li></ol>胚の3。機械的血管損傷<ol><li>転送ピペットを用いて、解剖実体顕微鏡でうつ病のスライドに麻酔をかけた胚を転送します。 </li><li>利き手で胚を操作するために注射針の短い平らな面を使用して、腹面が針から離れて向けてその側に胚を配置する。 </li><li>先端で特徴点のピンの位置を決め泌尿生殖器の開口部に魚の後部の腹面に対して直接指摘した。湾曲した先端が尾静脈（ 図1 &lt;に直接周皮を貫通することができるようにわずかな角度で特徴点のピンを配置します/強い&gt;）。 </li><li>胚を操作するために注射針を使用して、少し静脈にピンをフックするピンに胚をタップして特徴点ピンと尾静脈を貫通。 </li><li>注射針を使用して、容器内の小さな涙を作成するために離れて特徴点ピンから胚を引き出します。 注：成功した損傷は静脈から即時の出血になります。 </li></ol>止血4.分析<ol><li>目に見えて、この手順のために血液細胞の循環を持つ唯一の胚を選択してください。 </li><li>とすぐに特徴点ピンが容器から引っ張られるようにタイマーを開始する準備をします。 </li><li>とすぐに血液損失を傷から可視化することができるようにタイマーを起動します。傷口からの血液損失がなくなると、タイマーを停止し、出血時間として総時間を記録する。凝固が抑制される場合には、もはや目に見えて循環する血液細胞が存在しないときまでの時間を記録する。 </li></ol>創傷治癒の5.分析<ol><li>転送POS顕微鏡用ガラスボトムイメージング皿上にT-傷害動物。 </li><li>卵の水の大部分を削除します。 </li><li>アガロース42〜45ºCに加熱し、卵の水に溶解し、0.02％トリカインで補充0.3から1.2パーセントの低融点で胚をカバーする。 </li><li>ピンセットを用いて、その両側に位置胚。 </li><li>アガロースが冷却した後、卵の水中の0.02％トリカインで料理を埋める。 </li><li>明視野、落射蛍光、または共焦点顕微鏡を用いて画像を取得する。 </li><li>ピンセットを用いてアガロースから胚を取り出し、卵の水に戻って転送する。 </li></ol>

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The authors declare that they have no competing financial interests.

ゼブラフィッシュは、レーザー傷害13-15、レーザー誘発血栓16、および上皮創傷10を含む創傷の異なるタイプのモデルとして成功裡に使用されている。我々は、実行が簡単であり、リアルタイムの顕微鏡検査に非常に適しているin vivoモデルで制御怪我を生産機械的損傷の方法を報告している。迅速かつ測定可能な止血応答映像やタイムラプス顕微鏡を用いてモニ...

Zebrafish have been used extensively to study a variety of topics in vascular biology, including vascular development, angiogenesis, and hematopoietic development and pathology1-3. Embryos develop a functional circulation as well as leukocytes and other components of the innate immune system by 1 day post fertilization (dpf) 1,4,5. The conservation of the inflammatory and leukocyte response to injury has made the zebrafish embryo an informative model for such diverse inflammatory processes as tuberculous infection, enterocolitis, and tissue regeneration6-9. Zebrafish embryos have been used to study injury-related inflammation particularly in the context of epithelial wounding and the neutrophil response10,11. Injury to the embryo results in a highly conserved cellular response from cells at the injury site and the innate immune cells recruited to respond to the injury and regulate its resolution11,12. Other injury models have used focused laser pulses to spatially localize injury to specific cell types including neurons, muscle cells, and cardiomyocytes13-15. 
Zebrafish embryos have been used as a model to study hemostasis and thrombosis in conditions of pharmacological and genetic manipulation, using both mechanical and laser-induced thrombus formation16-19. Components of the coagulation cascade appear to be well-conserved and transgenics have allowed for detailed studies of thrombocyte and fibrin deposition at the site of coagulation17,20,21. The procedure presented in this paper complements these methods by providing a system for studying mechanical vessel injury resulting in vessel breach, thrombus formation and resolution, and vessel repair.

<table><tbody><tr><td>Minutia Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>Tip diameter 0.0125 mm, rod diameter 0.1 mm</td></tr><tr><td>Pin Holder</td><td>Fine Science Tools</td><td>26016-12</td><td></td></tr><tr><td>Dumont #5 Fine Tip Forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Glass Depression Slide</td><td>Aquatic Eco-Systems</td><td>M30</td><td></td></tr><tr><td>Low Melting Agarose</td><td>Lonza&amp;nbsp;</td><td>50081</td><td>Preheated to 42 &amp;ordm; C</td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma Aldrich</td><td>P7629</td><td></td></tr><tr><td>3-aminobenzoic acid (Tricaine)</td><td>Sigma Aldrich</td><td>E10521</td><td></td></tr><tr><td>Hirudin</td><td>Sigma Aldrich</td><td>H7016</td><td></td></tr><tr><td>Glass bottom imaging dishes</td><td>Mattek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZH10</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axio Observer</td><td></td></tr><tr><td>Aquarium salts</td><td>Instant Ocean</td><td></td><td></td></tr><tr><td>Insulin syringe with 28.5 G needle</td><td>Becton Dickinson&amp;nbsp;</td><td>329461</td><td></td></tr></tbody></table>

mechanical vessel injury in zebrafish embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

機械的な血管損傷が2 dpfの胚（ - C図2A）で行った。 （ 図2D）出血の停止までの時間によって測定されるように、迅速かつ信頼性の高い凝固応答の傷害をもたらす。凝固応答の差異を測定することができたか否かを判断するために、抗凝固ヒルジンはのために（直前に（水に溶解μlのヒルジンあた​​り1単位の5~10オランダ）を創傷にキュビエのダクトへの注入によっ?...

Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

Mechanical Vessel Injury in Zebrafish Embryos

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

ゼブラフィッシュ胚における機械的血管損傷

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

mechanical-vessel-injury-in-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

7.2K ビュー.  University of California. This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy. 

ビデオ: Mechanical Vessel Injury in Zebrafish Embryos

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

ゼブラフィッシュ胚におけるマイクロビーズの移植

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

発生生物学

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

Cryoinjuryを使用した大人のゼブラフィッシュにおける心筋梗塞の誘発

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

医学

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

心筋機械変形(DIAMOND)の変位解析は、胚性ゼブラフィッシュにおける心機能のセグメント的不均一性を明らかにする

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

神経科学

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

ゼブラフィッシュ胚の簡易かつ効果的な移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

<meta charset="utf8"></head><body>ゼブラフィッシュ心臓における磁気ビーズを用いたin vivoメカノトランスダクション研究

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction and physical stimuli generated by the beating heart. Intracardiac hemodynamics, driven by blood flow, emerge as fundamental information shaping the development of the embryonic heart. Here, we describe an effective method to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead in the cardiac lumen. The insertion of the bead is conducted through microsurgery in anesthetized larvae without perturbing heart function and enables artificial alteration of the boundary conditions, thereby modifying flow forces in the system. As a result, the presence of the bead amplifies the mechanical forces experienced by endocardial cells and can directly trigger mechanical stimulus-dependent calcium influx. This approach facilitates the investigation of mechanotransduction pathways that govern heart development and can provide insights into the role of mechanical forces in cardiac valve morphogenesis.

<meta charset="utf8"></head><body>著者スポットライト:ゼブラフィッシュの心臓の形成に関与する機械的な力を理解する

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

磁気ビーズを用いたゼブラフィッシュの心臓部における機械的な力の操作

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

生物学

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

イメージングおよび胚ゼブラフィッシュにおける脳血管構造物の3次元再構成

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

ゼブラフィッシュ胚における機械的血管損傷

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ゼブラフィッシュ胚におけるマイクロビーズの移植

Cryoinjuryを使用した大人のゼブラフィッシュにおける心筋梗塞の誘発

心筋機械変形(DIAMOND)の変位解析は、胚性ゼブラフィッシュにおける心機能のセグメント的不均一性を明らかにする

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

ゼブラフィッシュ胚の簡易かつ効果的な移植装置

磁気ビーズを用いたゼブラフィッシュの心臓部における機械的な力の操作

磁気ビーズを用いたゼブラフィッシュの心臓部における機械的な力の操作

磁気ビーズを用いたゼブラフィッシュの心臓部における機械的な力の操作

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

イメージングおよび胚ゼブラフィッシュにおける脳血管構造物の3次元再構成