Name: adult zebrafish injury models to study the effects of prednisolone in regenerating bone tissue
Uploaded: 2018-10-18T14:30:00
Description: Watch this Scientific Journal Video about Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue at JoVE.com

这项研究得到了德累斯顿再生疗法中心 ("斑马鱼作为一种模型来解开糖皮质激素引起的骨质流失的机制") 的资助, 另外还获得了德意志科技 (Transregio 67, 项目387653785) 至 FK。我们非常感谢1月卡斯林和 Avinash Chekuru 为他们的指导和协助钻孔引流 calvariae 和骨折的骨鳍射线。实验由 KG 和 FK 设计、执行和分析。FK 写了手稿。我们还要感谢卡瑞安兰伯特, 妮可 Cudak, 和其他成员的钮和品牌实验室的技术援助和讨论。我们也感谢玛丽卡费舍尔和依奇塔·福勒·弗兰科娃米希林为优秀的鱼护理和亨利埃特瞪大钮和乔希为校对手稿。

此处描述的所有方法均由德累斯顿 Landesdirektion 批准 (允许编号: az 24 d-9168.11-1/2008-1, az 24-9168.11-1/2011-52, az 24-9168.11-1/2013-5, az 24-9168.11-1/2013-14, az DD24.1-5131/354/87)。
1. 材料和解决方案的准备
注: 泼尼松, 像其他糖皮质激素, 导致免疫抑制。因此, 必须采取预防措施, 以防止在实验过程中被处理的动物感染。为此, 在开始实验之前, 热压罐玻璃器皿和 "鱼水" (即</em...

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斑马鱼在骨骼研究中被证明是有用的在许多方面。所选突变体模仿人类疾病的某些方面, 如成骨不全症或骨关节炎23、24、25、26、27和幼虫以及鳞片被用来在小分子筛中识别骨合成代谢化合物7,28,29。在临床...

斑马鱼是研究脊椎动物发育和疾病的强大动物模型。这是由于他们是小动物, 繁殖得非常好, 并且他们的基因组是完全排序和服从操作1。其他优势包括在不同阶段执行持续实时成像的选项, 包括成人斑马鱼2的活体成像, 以及在斑马鱼幼虫3中执行高通量药物筛查的能力。此外, 斑马鱼在包括骨骼在内的各种器官和组织中具有很高的再生能力, 因而成为研究骨骼疾病和修复4、5的有用系统。
糖皮质激素引起的骨质疏松症是一种由糖皮质激素长期治疗导致的疾病, 例如哮喘或类风湿性关节炎等自身免疫疾病治疗过程。在大约30% 的糖皮质激素治疗患者中发展, 并代表一个主要的健康问题6;因此, 深入研究免疫抑制对骨组织的影响是非常重要的。近年来, 开发了各种斑马鱼模型来处理其发病机制。糖皮质激素介导的骨丢失已被诱导在斑马鱼幼虫, 例如, 导致反作用化合物的鉴定增加在药物屏幕7的骨量。此外, 在斑马鱼鳞片的体外和体内8,9, 糖皮质激素诱导的骨抑制作用被模仿。这些检测是非常令人信服的方法, 特别是当它涉及到识别新的免疫抑制剂和骨合成代谢药物。然而, 他们只是部分考虑到内骨骼, 并没有在再生环境中执行。因此, 他们不允许调查糖皮质激素介导的作用在成人, 再生骨形成的快速模式。
在这里, 我们提出了一个协议, 使研究人员能够研究糖皮质激素介导的对成人斑马鱼骨骼再生的影响。损伤模型包括斑马鱼尾鳍的部分截肢, 头骨的钻孔引流, 以及翅片射线骨折的创建 (图 1A-1C), 并结合糖皮质激素暴露通过孵化 (图1E).我们最近使用了本协议的一部分来描述暴露于泼尼松 (通常规定的皮质类固醇药物之一) 对成人斑马鱼再生鳍和颅骨骨10的后果。在斑马鱼中, 泼尼松在单核细胞/巨噬细胞谱系10中导致成骨细胞增殖减少、成骨细胞分化不完全和细胞凋亡快速诱导。在本议定书中, 我们还描述了如何将骨折引入单个骨翅片11, 因为这种方法在研究骨折修复过程中的糖皮质激素介导的影响时可能很有用。这里介绍的方法将有助于进一步解决在快速再生的骨骼中的糖皮质激素作用的基本机制, 也可能被用于在斑马鱼组织再生的背景下系统药物管理的其他设置。

<table>
	<tbody>
		<tr>
			<td>Prednisolone</td>
			<td>Sigma-Aldrich</td>
			<td>P6004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxid (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl-3-aminobenzoate methanesulfonate (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>Aesculap</td>
			<td>BD027R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Braun</td>
			<td>5518059</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>840004</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle (0.3x13 mm)</td>
			<td>BD Beckton Dickinson</td>
			<td>30400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro drill</td>
			<td>Cell Point Scientific</td>
			<td>67-1000</td>
			<td>distributed e.g. by Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Steel burrs (0.5 &#181;m diameter)</td>
			<td>Fine Science tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Artemia ssp.</td>
			<td>Sanders</td>
			<td>425GR</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette (plastic, Pastette)</td>
			<td>Alpha Labs</td>
			<td>LW4111</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red S powder</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue 8 GX</td>
			<td>Sigma-Aldrich</td>
			<td>A5268</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T7409</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16 FA</td>
			<td>with QIMAGING RETIGA-SRV camera</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td>with Olympus DP71 or DP80 camera</td>
		</tr>
	</tbody>
</table>

adult zebrafish injury models to study the effects of prednisolone in regenerating bone tissue

斑马鱼能够在截肢后再生各种器官, 包括附属物 (鳍)。这涉及到骨头的再生, 这重生在受伤后大约两周内。此外, 斑马鱼能够在颅骨钻孔引流后迅速愈合骨骼, 修复可以很容易地引入斑马鱼骨鳍射线的骨折。这些损伤分析是测试药物对快速形成骨的作用的可行实验范式。在这里, 我们描述了这3损伤模型的使用和他们联合使用与系统性糖皮质激素治疗, 这会发挥骨抑制和免疫抑制作用。我们提供了如何准备成人斑马鱼免疫抑制剂治疗的工作流程, 说明了如何执行翅片截肢、颅骨骨钻孔引流和翅片骨折, 并描述了糖皮质激素的使用如何影响骨形成成骨细胞和单细胞/巨细胞谱系作为先天免疫的一部分在骨组织。

本协议在斑马鱼鳍和颅骨10、11、16的再生过程中反复使用, 以诱导快速骨形成。结合泼尼松的方法, 研究了泼尼松在骨再生中的作用。例如, 可以对再生过程中的骨形成和成矿作用进行研究。泼尼松, 作为其他糖皮质激素19,20, 导致全面抑制鳍再生, 包括骨形成, ...

Watch this Scientific Journal Video about Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue at JoVE.com

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

在这里, 我们描述了3成人斑马鱼损伤模型及其与免疫抑制药物治疗的联合使用。我们为再生组织的成像和在其中检测骨矿化提供指导。

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

这种方法可以帮助回答免疫抑制治疗干预领域的关键问题，用于研究药物对组织再生的不利影响，特别是对骨骼的不利影响。这项技术的主要优点是，它结合了过去的斑马鱼骨再生模型与全身药物暴露，将受伤的斑马鱼浸入药物补充鱼水中。在开始手术之前，每只斑马鱼用一个铝箔覆盖600毫升烧杯和适当数量的鱼水玻璃瓶进行实验，并在感兴趣的实验控制解决方案中准备免疫抑制药物。对于截肢伤害，使用钝钳小心地将麻醉的斑马鱼放在其侧侧 100 毫米 Petri 盘的内侧盖子上，并使用手术刀对 50% 的鳍进行修复。然后将鱼放入含有300毫升鱼水的自 <3> <3>化烧杯中，并辅以适当的实验剂，用铝箔盖住烧杯，防止斑马鱼逃跑。为了诱发鳍骨折损伤，在解剖显微镜下将麻醉斑马鱼放在其侧侧100毫米的加罗斯涂层培养皿中，然后将注射针稍微推入骨鳍射线段，直到出现裂纹。重要的是，在鳍中引入不要太多的骨折，因为这可能会极大地损害稳定性。然后将鱼转移到含有300毫升鱼水的自用烧死嘴中，并辅以适当的实验剂。要产生钙化颅骨损伤，请将麻醉的斑马鱼放在直立位置，以便钙骨在解剖显微镜下很容易可视化。将装有 500 微米钻头的旋转微钻头放在前骨中心，轻轻触摸骨骼，生成一个微型钻孔大小的孔。在产生损伤时不要横向移动钻头，一旦组织阻力下降，立即停止钻头至关重要。否则，大脑就会受损。然后将鱼放入含有300毫升鱼水的自动捕获的烧杯中，并辅以适当的实验剂。每天将斑马鱼和鱼水转移到适当的临时容器中，用新鲜药物补充鱼水重新灌装烧杯，改变烧杯中的水。然后用鱼网将斑马鱼还到烧杯上。对于总共持续两天的治疗，不要喂斑马鱼。在较长的实验过程中，每两天用0.5至1毫升孵化的阿提米亚SSP喂养斑马鱼。对于鳍伤害分析，收获细，并使用细钳抓住鱼翅在树桩的一个边缘。使用第二对钳子抓住相反的树桩边缘，然后稍微将组织压在含有冷 4%PFA 的盘子底部 10 到 20 秒。根据需要重复鳍展平。鳍现在应该平放，没有卷曲。对于固定后的长期样品储存，用三个20分钟的PBS洗涤和上升的甲醇系列清洗组织。然后，样品可以储存在零下20摄氏度的100%甲醇中。对于Alizarin Red染色，在降压甲醇系列中储存补水甲醇，随后在PBS中洗涤20分钟，在去维化水中清洗5分钟。最后一次洗涤后，将Alizarin Red溶液加入样品组织，并在室温下用摇动孵育样品，以进行适当的染色期。要清除样品，用减少的1%氢氧化钾甘油系列处理组织。然后将样品储存在一对一的 0.1% 氢氧化钾溶液中，然后用 80% 甘油进行组织安装，以进行想象。对于 Calcein 染色，在用铝箔覆盖的玻璃烧杯中同时孵育多达三条斑马鱼，在 100 毫米的钙素溶液中孵育 20 分钟。在孵化结束时，将斑马鱼转移到鱼水的容器中，让鱼短暂游泳，然后再将它们转移到鱼水的新容器中。在第二个烧杯20分钟后，获得鱼翅再生的实时图像，将麻醉斑马鱼放在一个加糖涂层的培养皿上，并通过立体显微镜对鳍进行成像。要获取再生头骨的图像，请将斑马鱼直立在海绵中，然后通过荧光显微镜对头骨进行成像。与其他糖皮质激素类似，使用 Prednisolone 进行治疗，可对鳍再生进行整体抑制，包括由 Alizarin Red 染色的固定果鳍组织检测到的骨骼形成。同样，这种药物对颅骨损伤闭合有延迟作用。由于免疫抑制，减少巨噬细胞数也可以通过冷冻组织部分的免疫组织化学检测，如使用抗mCherry和抗GFP抗体染色和转基因MPEG-1 mCherry与Osterix NGFP斑马鱼交叉在斑马鱼头骨组织样本从前尼索酮治疗的动物。在尝试这些程序时，重要的是通过以高度可重复的方式执行损伤论文，以尽量减少骨再生速度的变异性。在含有自体鱼水的自体嘴鱼中，单屋斑马鱼也至关重要，以避免微生物感染。该协议将有助于进一步解决糖皮质激素作用的基本机制。例如，在糖皮质激素诱导骨质疏松症的背景下，也可以适用于其他药物和其他斑马鱼骨的使用。

Research

JoVE Journal

Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

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 ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

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 ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Live Imaging of Mouse Secondary Palate Fusion

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...