我们要感谢特伦特沃他的技术援助。我们也承认儿科系病童医院和治疗先天性肌营养不良（CMD）的慷慨资助这一项目。

1.准备琼脂注射板（时间:45分钟） <ol><li>煮2％至3％琼脂糖在E3媒体和允许的解决方案，以在板凳上略有降温。注意:正在准备注射板的数量表明琼脂糖的需要量。每次注射板需要约35毫升琼脂糖溶液中。 </li><li>煮沸后，使琼脂糖冷却直到达到所需的温度（例如，45℃），为每注射模具制造商的说明。 </li><li>倒入约35毫升冷却的琼脂糖为100毫米的菜。 </li><li>将首选注塑模具到溶液中的一端，然后躺在模具的其余部分到琼脂糖溶液（这将有助于减少气泡的产生）。 </li><li>允许琼脂糖溶液固化或者在RT或4℃大约30分钟。 </li><li>用刮刀到模具的一个端部从所述固体琼脂糖分离。慢慢地取出m个剩余<html>旧。 </li></ol> 2.准备埃文斯蓝色染料（EBD）注射液混合的（时间:30分钟） <ol><li>使EBD 1％的库存在1X林格氏液（155毫摩尔NaCl; 5mM的氯化钾; 2毫米氯化钙2; 1mM的MgCl 2的; 2毫的 Na 2 HPO 4; 10mM的HEPES; 10mM葡萄糖; pH至7.2），它可以储存在室温。 </li><li>使异硫氰酸荧光素储备溶液（FITC） -葡聚糖分子量10000 kDa的25毫克/毫升在1X林格氏溶液并储存在-20℃。 </li><li>准备注射液混合在了FITC葡聚糖股票原液稀释EBD直接0.1％（即，对于最终工作100微升量:将10微升1％EBD的90微升FITC葡聚糖股）。 </li><li>彻底涡流喷射混合（它应该变为绿色），远离直射光通过包装注入混合管铝箔。 </li></ol> 3. EBD注射剂（时间:约30分钟） ENT"&gt;注:协议在最佳状态下3-7天受精后（DPF）的幼虫。 <ol><li>前期预热注射板RT。 </li><li>设置注射装置通过布置显微上的金属板和站旁的显微镜被用于注射。打开空气驱动显微注射控制器。注:优选的注射系统将通过实验室变化，并且不应该改变的分析结果。 </li><li>回填土注射针，大约2-4微升EBD组合。 </li><li>校准注射量为约5 NL EBD组合。注意事项:注射量校正将取决于校准方法。活塞驱动的注射可以直接设置到给定的注射量而气体压力喷射器将需要的注射体积通过量丸药与使用千分尺校准。 </li><li>湿式喷射板1X林格氏液，并从井中取出多余的。 </li><li>预治疗幼虫用0.04％的3-氨基苯甲酸乙酯甲磺酸盐萨尔吨（三卡因）稀释在1X林格氏液来固定在注射前的起始幼虫。注:确保幼虫完全immotile是重要的，因为正确的注射液是很难与任何残留的运动。 </li><li>放置麻醉幼虫，并将使用的玻璃吸移管的琼脂喷射板的孔。确保幼虫完全井内，躺在自己的身边。注意:每孔幼虫的数目是高达实验者。 </li><li>被放入孔后幼虫，除去过量的林格氏液在井内最小化幼虫运动。留下的溶液的残留量，以使得幼虫不脱水。 </li></ol>斑马鱼幼虫4.心包注射EBD（时间:取决于幼虫注射的数量，估计1-3个小时） <ol><li>上放置装有其中注射将执行解剖范围幼虫注入板。 </li><li>定位包含注射针针该EBD混合了斑马鱼幼虫。 </li><li>重新定位的注入板通过旋转它，以便将注射针靠近幼虫的心脏和大约45°腹侧从前后轴。 </li><li>将注射针头插入公共主静脉（CCV）在静脉的区域的蛋黄，其中静脉最初转动在背方向（图1）的前部。注:最高40倍的放大倍率可能有助于清楚地看到CCV。 </li><li>注入5 NL EBD组合，并保持注射针在位置5-8秒，以尽量减少直接泄漏EBD组合。注意:一个好的注射将有染料着色见于心脏腔室（ 图1）。如果EBD混合物中未观察到心脏，然后注入额外的5 NL EBD混合物可以是足以诱导染率。可替代地，胚胎可以被丢弃。 注:在某些情况下，心脏会停止跳动。如果这Øccurs，继续监视幼虫20-40秒。通常情况下，心脏跳动恢复作为染料通过循环系统移动。 </li><li>进入下一个幼虫和重复。 </li><li>通过注射（图2）后，立即观察的FITC-葡聚糖在脉管的存在识别成功注射的胚胎。 </li></ol> 5.孵化和EBD吸收（时间:4-6小时） <ol><li>后幼虫的期望数量的注入，返回在100mm平皿注入幼虫1X林格氏液无三卡因。 </li><li>保持菜肴用铝箔包裹。注意:保持注射幼虫在黑暗中显著提高生存率，并确保在信号强度最大的一致性。包裹在铝箔为的时间段，该幼虫的保育箱的外面尤其重要。 </li><li>允许幼虫孵化在28.5℃，4-6小时，以保证有足够的EBD摄取。 </li></OL&gt; 6，可视化EBD的肌肉（时间:取决于幼虫注入和显微镜的类型的数量，预计0.5-3小时） <ol><li>在成像前，麻醉幼虫用0.04％三卡因以防止移动。 </li><li>下红色荧光视图幼虫以确定是否EBD摄取是发生在骨骼肌（图3）。 </li></ol>

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作者有没有竞争经济利益或其他的利益冲突。

斑马鱼正在成为一个强大的工具，用于神经肌肉疾病2,29研究。迄今为止，斑马鱼系统已用于验证新的肌肉致病突变16,17,30，阐明新颖病理机制18，并识别潜在的新的治疗药物12,24。这些集体努力已经建立了斑马鱼的实用程序来模拟人类神经肌肉疾病。然而，尽管与斑马鱼和哺乳动物模型所取得的进展，有用于神经肌肉条件宽光谱内患者有限的治疗选择。因此，一个高需求存在治疗的发展，这组毁灭性的疾病。并联的治疗这种需求对应的需要不断实验创新，以及严谨的分析，以验证新的动物模型和假定的治疗策略。 EBD分析是常用的小鼠模型来研究组织和在脑，心脏细胞损伤，和骨骼肌27,31。最值得注意的是，EBD被广泛应用于各种肌营养不良症亚型的小鼠模型显示的肌膜不稳定的严重性和损坏8。使用EBD的揭示肌肉膜损伤是一个有利的参数建立动物模型的相似性对人类疾病状态9。 EBD在鼠标的电源已导致几个实验室，包括我们自己，开发和应用EBD神经肌肉疾病的斑马鱼模型。由于EBD分析的适用性，这种技术正在积极实施以证实斑马鱼的模型与人类疾病状态11,15,22,24,32。幼虫受损的肌肉的膜将具有肌纤维内EBD摄取，因此红色荧光。荧光在纤维间空间观察到的，但不是个别的肌肉纤维中也可以是信息性的纤维，从在吨基底膜分离的他没有膜的损伤，提供有用的诊断细节。 EBD分析已经超越动物模型验证的应用前景。从我们的实验室努力最近证明，EBD分析，有利于在确认潜在的新型治疗药物24。如果确定潜在的治疗治疗减少或取消EBD摄取神经肌肉疾病模型可以表示相关的治疗作用8。这种类型的分析可以帮助确定治疗的机制（S）和扩展EBD分析中的应用。 与许多技术，EBD分析确实有几个注意事项在实验设计和实践中加以观察。例如，它可以是具有挑战性的识别CCV由于随着年龄的组织的增厚。此外，它很容易损坏幼虫在制备之前和心包注射期间，减少实验计数和增加的需要来准备大量的幼虫。此外，处理和注射过程中所做的幼虫物理损坏可能会导致误报受损的肌肉会占用EBD。为了克服这些障碍，我们这个视频文章，它允许容易和可靠识别幼虫与成功的染料输注后立即注入和之前随后的分析中所描述的共注入策略。的FITC-葡聚糖共同注射对照成功注射通过允许在脉管确认EBD的之前，其吸收的肌肉纤维。作为EBD荧光变得在幼虫高度扩散，如果在肌肉纤维未收集几个小时后这可以是特别有用的;因此，它可能难以察觉。此外，缺少CCV和注入EBD入蛋黄或体腔可以培养后，导致弥漫性红色荧光类似于控制胚胎，但有吸收的受损的肌肉纤维的可能性降低。总的来说，这些洞穴ATS建议EBD注射需要耐心和实践，以达到一致和可靠的结果。 总之，我们描述进行EBD分析斑马鱼幼体实际和直接的方法。迄今为止，使用斑马鱼作为模型系统，尤其是作为人类疾病模型，已迅速扩大。这种扩张部分是由于改善后的斑马鱼系统的当前优势的实验技术的不断发展和改进。该EBD注射技术提供了一个额外的，功能强大的工具，以研究人员的武器库的验证和斑马鱼肌肉疾病模型的研究。这种技术的不断执行和修改有帮助发现新的治疗策略，以及引起疾病机制的潜力。

Muscular dystrophies constitute a group of prevalent and heterogeneous human muscle diseases with specific histopathological features1,2. Symptoms typically associated with this devastating group of diseases include muscle weakness, muscle degeneration, loss of motility, and early mortality1,3. The primary pathomechanisms of muscular dystrophies are the loss of proteins that stabilize the sarcolemma, anchor transmembrane complexes, and mediate cell signaling across the membrane4-6. For example, complete loss of the protein dystrophin, a primary scaffold protein of the dystrophin-glycoprotein complex, results in destabilization of the muscle membrane in Duchenne muscular dystrophy7. Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity8,9. This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems2,10-15. However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes. This limitation of viable therapies is due to several key factors: 1) the difficulty of gene therapy strategies, 2) a high frequency of de-novo disease cases and the corresponding lack of translatable animal models, and 3) the lack of rigorous strategies to test the physiological consequences of putative therapeutic agents with clear and reproducible outcome measures.
To overcome some of these limitations, numerous labs including our own are employing zebrafish as a system to model and study human neuromuscular diseases2. To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations16,17, elucidate uncharacterized disease causing mechanisms17,18, and identify novel therapeutic strategies12,19. These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers20. Zebrafish have additionally proven amendable to large-scale drug screens21, a valuable method for the identification of novel therapeutics22-24. Regarding muscle disease research, these strengths are complemented by the ability to isolate single zebrafish skeletal muscle fibers via dissociation25 and by the ability to examine myofiber organization in vivo using the optical phenomenon called birefringence26, which collectively allows for rapid determination of macroscopic muscle integrity. Regardless of these available utilities, further tool development is continuously required to advance investigation.
We, and others, have adapted a protocol for EBD injection and analysis in the zebrafish model. EBD is a vital, cell permeable dye that is taken up by damaged, degenerating, or apoptotic cells and then visualized under fluorescence27. To date, EBD analysis has extensively been used to analyze muscle membrane integrity in mouse models of skeletal muscle and heart diseases8,9,27. However, in mammalian preparations, harvested muscle typically requires laborious sectioning or dissection prior to analysis. In zebrafish, direct analysis is possible in high numbers using live and intact animals. In this video article, we will demonstrate the methodology to perform EBD injection and analysis in live zebrafish larvae, with representative images of EBD uptake in the zebrafish dystrophy mutant line sapje15,28. Furthermore, we present a co-injection strategy that allows for increased quantification of EBD preparations.

<table><tbody><tr><td>Fluorescein isothiocyanate-dextran MW 10,000</td><td>Sigma</td><td>FD10S</td><td></td></tr><tr><td>Evan&#039;s Blue Dye</td><td>Sigma</td><td>E2129</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>100 mm Petri dish</td><td>Fischerbrand</td><td>FB0875712</td><td>Injection mold base</td></tr><tr><td>Thin wall glass capillaries</td><td>World Precision Instruments</td><td>TW100F-4</td><td>For Injection needle</td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA001</td><td>Injection mold</td></tr><tr><td>Microinjection mold</td><td>Adaptive Science Tools</td><td>TU-1</td><td>Injection mold</td></tr><tr><td>Sodium chloride</td><td>Bioshop</td><td>SOD001</td><td>Ringer&#039;s solution</td></tr><tr><td>Potassium chloride</td><td>Bioshop</td><td>POC888</td><td>Ringer&#039;s solution</td></tr><tr><td>Magnessium chloride hexahydrate</td><td>Sigma</td><td>M2670</td><td>Ringer&#039;s solution</td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td>Ringer&#039;s solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>Ringer&#039;s solution</td></tr><tr><td>Glucose</td><td>BioBasic</td><td>GB0219</td><td>Ringer&#039;s solution</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C1061</td><td>Ringer&#039;s solution</td></tr></tbody></table>

analysis of zebrafish larvae skeletal muscle integrity with evans blue dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

所述EBD注射混合物在3旦注入的sapje纯合突变体和野生型同胞的CCV。注射弥漫于心脏腔室（图1B），然后成功注射通过可视化FITC-葡聚糖在根据绿色荧光脉管（图2）进行分析。 经过4小时的潜伏期，EBD摄取检查在用荧光显微镜体节的水平。野生型同胞内表现出任何可见的肌纤维（图3A）没有EBD荧光，而纯合sapje突变体显示EBD摄取，指示损坏肌膜15（图3B）。 <img alt="图1" src="/files/ftp_upload/53183/53183fig1.jpg" /> 图1.注射EBD注入搅拌成斑马鱼安莉芳的共同主静脉（CCV）O操作。 （一）未注射胚胎。箭头表示的CCV注射理想地点。 （二）成功注入CCV。染料进入心脏腔室（箭头），并开始通过脉管被泵送。 （C）不成功的CCV注入将导致部分或全部进入胚胎（箭头）的卵黄囊的染料。 <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53183/53183fig2.jpg" /> 图2.胚胎可以通过观察在绿色荧光FITC-葡聚糖分布于整个脉管紧跟的喷射和前EBD摄取被排序为成功的注射。请点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53183/53183fig3.jpg" /> 图3:EBD将采取由受损的膜纤维（A）野生型的兄弟姐妹在展示肌肉纤维没有EBD荧光。 （二）Sapje ​​纯合突变与多个肌纤维（箭头）内EBD荧光。所有幼虫注射了EBD注射混合，并在3旦一个4小时的潜伏期后进行分析。兄弟姐妹和突变体是由前CCV注射肌纤维脱离排序。 <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

analysis-zebrafish-larvae-skeletal-muscle-integrity-with-evans-blue

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  The Hospital for Sick Children. In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

视频: Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

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

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

采用触摸诱发反应和运动测定以评估在斑马鱼肌肉性能和功能

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

科研

发育生物学

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

建立有限元模型，研究斑马鱼下颌生物力学

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

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

使用茜素红染色检测斑马鱼幼虫中化学诱导的骨质流失

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

生物学

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...