对于侧线再生成年斑马鱼的含量
Summary
由于神经和非神经系统疾病的许多斑马鱼模型研究在成年鱼，而不是胚胎/幼虫，我们开发了可应用于成年斑马鱼疾病模型的定量侧线再生试验。该法涉及的分辨率在1）神经丘和2）单个毛细胞的水平。
Abstract
由于听力和平衡紊乱在人的临床重要性，模式生物如斑马鱼已被用于研究横向线的开发和再生。斑马鱼是由于其快速的开发时间和它的高再生能力为这些研究特别有吸引力的。迄今为止，侧线再生斑马鱼的研究主要是利用，因为在这几个阶段的较小的数字神经丘的胚胎和幼虫的鱼。这使侧线再生/和或更早发育阶段的开发更容易进行定量分析。由于神经系统和非神经系统疾病的许多斑马鱼模型研究了成鱼，而不是在胚胎/幼虫，我们专注于开发一个定量的侧线试验再生的成年斑马鱼，这样的分析是可用的，可以被应用到当前成年斑马鱼疾病模型。由Van TRUM建立在前人研究所描述的毛细胞在成年墨西哥洞穴盲鱼和斑马鱼（ 斑马鱼 ）的消融过程P 等17中 ，我们分析的目的是让控制组和实验组之间的定量比较。这是通过开发一种基于神经丘再现过以下毛细胞在限定的横向线的区域庆大霉素引起的坏死一个24小时时间段的百分比的再生神经丘标准曲线来实现。该测定也被设计为允许扩展的分析，以当需要的分辨率更高水平的个体毛发细胞的水平。
Introduction
侧线（LL）系统是鱼类和两栖类，它负责听觉，平衡，rheotaxis调处行为，如教育和捕食者回避1-5找到了mechanosensory器官。这两者它是由簇毛细胞，支持细胞包围，被定位在结构，称为神经丘6。这些神经丘通常组织成垂直线（称为针）沿主体和尾部与一些在鱼的头部观察到的水平线圈的纵向轴线。在成人中，神经丘是显著更大数目的线圈相比，胚胎或仔鱼6内。在斑马鱼生物医学研究都集中在抗生素治疗，噪音引起的创伤，慢性感染等的影响。对毛细胞，试图7,8，以便更好地了解他们在人体的影响。
与大多数脊椎动物，德硬骨鱼，如斑马鱼（ 斑马鱼 ），必须重新生成丢失的毛细胞的能力。因为它们的快速开发时间和高再生能力的斑马鱼是特别有用的。迄今为止，然而;侧线上的开发和/或再生斑马鱼研究主要利用的胚胎和幼虫阶段的鱼，由于侧线神经丘的数量减少可以较容易计数和分析6,9,10。
然而，神经系统和非神经系统疾病11-16尽可能多的斑马鱼模型研究了成鱼，而不是幼虫，我们专注于开发在成年斑马鱼用庆大霉素横向线再生法（以前在斑马鱼幼虫用一种氨基糖苷类和最近与成年鱼17用），这样的测定法是可用的，可以被应用到当前的成年斑马鱼的疾病模型。而此前公布的程序由Van特朗普ét铝，17设立在成鱼的毛细胞消融的条件，他们没有建立标准曲线这是需要控制组和实验组利用转基因斑马鱼线或药物诱发的疾病状态时，如之间的定量比较神经丘再生在斑马鱼18。因此，我们跟着范·特朗普等人 17的毛细胞消融的程序，但建立在他们的工作，以建立神经丘再生的标准曲线，以使研究者比较对照组和实验组在使用我们的数据，如与成年斑马鱼疾病模型。该测定也被设计为允许扩展的分析对个体毛发细胞时，需要解决的一个更高的水平。
Protocol
所有的程序都按照“实验动物福利原则”所述的准则进行（卫生出版没有。85-23国家研究院，1985年修订）和经批准的罗莎琳德富兰克林大学实验动物管理和使用委员会动物协议08-19。 1，庆大霉素诱导的毛细胞坏死制备硫酸庆大霉素生理盐水在0.004％的终浓度（4.32毫摩尔）。 在含有0.004％（4.32毫米）庆大霉素溶液的容器放置成鱼（ 斑马鱼 ，4-6个月的…
Representative Results
优化的程序，在量化成年斑马鱼的侧线神经丘再生。 斑马鱼幼虫的神经丘是容易量化;然而，成年斑马鱼的侧线具有更大数目的每针脚神经丘作出定量分析更加困难6,17,19,20的。就像在图1A中 ，头部有许多神经丘相比，任一中间部分或尾部的显著更高;用具有如图所示的一维数最少的神经丘的尾部区域。因为线圈中的头…
Discussion
基于已建立的侧线（LL）再生的胚胎和幼虫斑马鱼8,24,25分析文献的广泛身体，我们的研究目标是开发在斑马鱼的定量检测横向线条的再生可能被应用到被研究最好在成年鱼的疾病模型。我们发现，应用到成鱼开发的胚胎/仔鱼过程时，某些关键点是很重要的。最重要的这些点被认为：1）侧线的数量沿鱼的纵向轴线神经丘，神经丘毛细胞染色的2），3）治疗的氨基糖苷的浓度和持续时间，和4…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors have nothing to disclose.
Materials
| Gentamicin sulfate solution (50mg/ml) | Sigma Aldrich | G1397 | |
| 2 Phenoxyethanol | Sigma Aldrich | P1126 | |
| 4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp) | Aldrich | D-3418 | 485 nm excitation λ and 603 nm emission λ |
| in methanol | |||
| 6 well plates | Mid Sci | TP92006 | |
| Petri Dishes | Fisher Scientific | 08-757-13 | |
| Glass Bottom Microwell Dishes | Matek Corporation | P35G-1.5-14-C | |
| Sodium Chloride | Sigma Aldrich | S3014 | |
| Dissecting Microscope | Nikon | TMZ-1500 | Any dissecting microscope is fine. |
| Camera for Imaging | Nikon | Q imaging | Any camera is suitable. |
| Image J software | National Institutes of Health | NIH Image | |
| NIS Elements | Nikon | Any imaging software is suitable. | |
| Confocal microscope | Olympus | FV10i | Any high resolution fluorescent microscope is suitable |
| Aquatic System | KG Aquatics ZFS Rack System. | Any aquatic system can be used |
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Citazione di questo articolo
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