斑马鱼幼虫骨骼肌诚信与伊文思蓝分析
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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。
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Fluorescein isothiocyanate-dextran MW 10,000 | Sigma | FD10S | |
| Evan's Blue Dye | Sigma | E2129 | |
| Ethyl 3-aminobenzoate methanesulfonate salt | Sigma | A5040 | |
| 100 mm Petri dish | Fischerbrand | FB0875712 | Injection mold base |
| Thin wall glass capillaries | World Precision Instruments | TW100F-4 | For Injection needle |
| Agarose | Bioshop | AGA001 | Injection mold |
| Microinjection mold | Adaptive Science Tools | TU-1 | Injection mold |
| Sodium chloride | Bioshop | SOD001 | Ringer's solution |
| Potassium chloride | Bioshop | POC888 | Ringer's solution |
| Magnessium chloride hexahydrate | Sigma | M2670 | Ringer's solution |
| Sodium phosphate monobasic monohydrate | Sigma | S9638 | Ringer's solution |
| HEPES | Sigma | H4034 | Ringer's solution |
| Glucose | BioBasic | GB0219 | Ringer's solution |
| Calcium chloride | Sigma | C1061 | Ringer's solution |
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