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:使用斑马鱼的程序批准了加州大学旧金山分校的机构动物护理和使用委员会。 1.准备工具<ol><li>将细节针入针座，钳制引脚。 </li><li>用细尖镊子，小心翼翼地弯针的针尖创建一个轻微的钩子。 </li><li>对于操作和伤害在胚胎稳定，弯曲28瓜哥½英寸针安装在使用尖嘴钳胰岛素注射器的结束。 </li></ol> 2.准备斑马鱼胚胎的损伤<ol><li>成立了斑马鱼的繁殖对收集的鸡蛋水（60ug / mL的水族馆盐）蛋之前22所示。 </li><li>添加0.003％N-苯基硫脲（PTU）的蛋水时的胚胎是大约8小时受精后（HPF），以防止黑化。 </li><li> Dechorionate2天受精后（DPF）用细尖镊子胚胎实验之前。 <br/&gt;注:循环开始后胚胎可以随时受伤。这里介绍的数据为2旦的胚胎，但该技术已经在胚胎中得到成功应用高达5单丝旦。 </li><li>麻醉用0.02％缓冲的3-氨基苯甲酸（三卡因）之前的操作约10分钟的胚胎。 </li></ol> 3.机械船舶胚胎损伤<ol><li>麻醉转移胚胎到抑郁症的幻灯片上用移液管解剖体视显微镜。 </li><li>使用注射器针头的短边平来操纵与优势手的胚胎，朝向远离针腹面定位在其一侧的胚胎。 </li><li>定位销细节与尖端直接指向对鱼后方泌尿生殖开口的腹面。定位销细节以一个很小的角度，使得所述弯曲端部能够通过周皮直接刺穿入尾静脉（ 图1 &lt;/ STRONG&gt;）。 </li><li>使用注射器针头操纵胚胎，通过敲击胚胎成销稍微钩销插入静脉刺穿尾静脉用细节销。 </li><li>用注射器针头，拉胚离细节引脚创建一个小的撕裂血管。 注:一个成功的损伤将导致从静脉直接出血。 </li></ol>止血4.分析<ol><li>有明显的循环血细胞在此过程中只选择胚胎。 </li><li>准备只要细节销从容器拉出开始定时器。 </li><li>只要血液损失可以从伤口被可视启动定时器。当从伤口失血停止，停止计时和总时间记录为出血时间。如果凝固被抑制，记录时间到时不再有明显的循环血液细胞。 </li></ol>伤口愈合5.分析<ol><li>传输POST-伤害动物到玻璃底菜成像显微镜。 </li><li>去除大部分的鸡蛋水。 </li><li>覆盖在0.3-1.2％低熔点胚胎琼脂糖溶解在蛋水，加热至42之间和45ºC和补充有0.02％三卡因。 </li><li>位置的胚胎在使用镊子身体两侧。 </li><li>琼脂糖冷却后，装满鸡蛋的水0.02％三卡因的菜。 </li><li>通过收购明，落射荧光或共聚焦显微镜图像。 </li><li>使用镊子从琼脂糖取出胚胎，并传回的鸡蛋水。 </li></ol>

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The authors declare that they have no competing financial interests.

斑马鱼已经成功地使用的一种模式的不同类型的伤口，包括激光损伤13-15，激光诱导的血栓16和上皮伤人10。我们报告机械损伤的方法，该方法是简单的执行，并在体内模型中是高度适合于实时显微镜产生一个受控的损伤。损伤产生快速和可测量的止血反应并且可以使用视频和缩时显微术来监测一个重现的伤口修复程序。 其简单和刻板血管解剖?...

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.

机械血管损伤2进行DPF胚胎（ 图2A - C）。损伤产生快速和可靠的电凝反应如通过时间测量以停止出血（ 图2D）的。以确定是否在凝固反应差异可以测量，抗凝血剂水蛭素施用给胚胎注射入居维叶的管道伤人（NL每微升水蛭素溶解在水中1单元5-10）之前立即（对于示范注射到居维叶的管道中，看到以前的朱庇特的第23条）24。水蛭素的前损伤导致?...

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.

在斑马鱼胚胎的机械血管损伤

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 次观看.  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. 

视频: 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.

微珠植入斑马鱼胚胎

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 ...

科研

发育生物学

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.

在成年斑马鱼心肌梗死的感应使用Cryoinjury

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.

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

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.

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

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

神经科学

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.

一种简单有效的斑马鱼胚胎移植装置

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>在斑马鱼心脏中使用磁珠的体内机械转导研究

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>作者聚焦:了解塑造斑马鱼心脏所涉及的机械力

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.

使用磁珠操纵发育中的斑马鱼心脏中的机械力

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.

激光伤害的斑马鱼胚胎骨骼肌

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.

成像和胚胎斑马鱼脑血管结构的三维重建

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

在斑马鱼胚胎的机械血管损伤

摘要

探索更多视频

此视频中的章节

微珠植入斑马鱼胚胎

在成年斑马鱼心肌梗死的感应使用Cryoinjury

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生

一种简单有效的斑马鱼胚胎移植装置

使用磁珠操纵发育中的斑马鱼心脏中的机械力

使用磁珠操纵发育中的斑马鱼心脏中的机械力

使用磁珠操纵发育中的斑马鱼心脏中的机械力

激光伤害的斑马鱼胚胎骨骼肌

成像和胚胎斑马鱼脑血管结构的三维重建

在斑马鱼胚胎的机械血管损伤

摘要

探索更多视频

此视频中的章节

微珠植入斑马鱼胚胎

在成年斑马鱼心肌梗死的感应使用Cryoinjury

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

一种简单有效的斑马鱼胚胎移植装置

使用磁珠操纵发育中的斑马鱼心脏中的机械力

使用磁珠操纵发育中的斑马鱼心脏中的机械力

使用磁珠操纵发育中的斑马鱼心脏中的机械力

激光伤害的斑马鱼胚胎骨骼肌

成像和胚胎斑马鱼脑血管结构的三维重建

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生