成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像
Summary
斑马鱼视网膜再生大多被使用固定的视网膜研究。然而，再生反应过程中发生的动态过程，如interkinetic核迁移，并需要活细胞成像研究的基本机制。这里，我们描述培养和成像条件下，使用多光子显微术来监控Interkinetic核迁移（INM）的实时性。
Abstract
内源性再生程序是由穆勒神经胶质细胞在成年斑马鱼的神经元以下的损伤和死亡发起（斑马鱼 ）视网膜。的米勒胶质细胞重新进入细胞周期并产生经过随后的几轮细胞分裂并分化成丢失的神经元细胞类型的神经元祖细胞。既米勒胶质细胞和神经元祖细胞的细胞核复制它们的DNA，并且在描述为Interkinetic过程视网膜， 即它们的基内核层（INL）和外核层（ONL）之间分别迁移，的不同的位置进行有丝分裂核迁移（INM）。 INM已主要研究了在显影视网膜。审查INM在成人再生详细斑马鱼视网膜的动力学，需要荧光标记的米勒胶质细胞/神经元祖细胞的活细胞成像。在这里，我们提供给隔离条件和文化背视网膜从Tg为GFAP：nGFP]认为暴露于恒定的强光35 H mi2004斑马鱼。我们还表明，这些视网膜文化是可行的执行活细胞成像实验，利用多光子显微镜来监测GFAP的迁移行为在整个视网膜外植体的厚度是连续获得Z堆栈图像长达8小时：nGFP阳性细胞。此外，我们描述的细节来执行后成像分析，以确定根尖和基底INM的速度。总而言之，我们制定的条件来研究INM的动力学在神经再生的成年模型。这将推动我们的这一关键细胞过程的了解，使我们能够确定控制INM的机制。
Introduction
与人类不同，斑马鱼（Danio rerio）显示出在视网膜神经元1，2，3，4的细胞死亡的健壮再生反应。肿瘤坏死因子α，即从濒死的视网膜神经元释放的信号传导分子诱导驻留在基底内核层的视网膜（INL）在米勒胶质细胞，增殖5并产生神经元祖细胞继续分化成神经元细胞之前增殖即死2种，3，4。在再生反应的增殖期，米勒胶质细胞的细胞核和其衍生的神经元祖细胞阶段进行的重复洄游图案与细胞周期（Interkinetic核迁移，INM）6 </sup >，7。定位在基底INL细胞核迁移到外核层（ONL），他们划分而产生的核基部返回到INL之前之前复制它们的DNA。这个过程的神经上皮发育期间利用组织学方法首先描述，而活细胞成像的方法后来被绍尔8，9，10，11，12证实了解释。两个组织化学和活细胞成像的方法已被用来确定底层INM及其发展神经上皮包括视网膜9，11，12，13的功能的机制。然而，管理在成人视网膜再生的INM机制尚未研究了很多细节外部参照“> 6，7，活细胞成像将推动我们在成人视网膜再生控制INM的信号通路知识的宝贵途径。
直到最近，在视网膜INM的活细胞成像限于任一活斑马鱼的胚胎或胚胎鸡或产后小鼠视网膜植9，10，11，12，14，15，16。而从各种物种的成年动物，包括小鼠，大鼠和斑马鱼视网膜植已被用于不同的细胞生物学方法17，18，19，20，利用视网膜植公顷活细胞成像实验已经被限制向大家介绍的时间段，并没有被数小时21,22连续执行。在这里，我们描述了一个详细的协议，以文化的光损伤成年斑马鱼视网膜利用多光子显微镜6进行活细胞成像实验监测INM。调查控制INM机制时，作为INM的动态， 例如，速度可能会受到影响，而不是有丝分裂的位置，这将潜在地不使用免疫细胞化学来检测活细胞成像的方法是通过免疫组织化学方法是有利的。
在将来，这种方法有也可能被修改，以研究视网膜再生期间其它动态过程，如垂死由米勒胶质或小胶质细胞的行为的光感受器的吞噬作用。
Protocol
注：斑马鱼中提出并维持在Freimann生命科学中心在巴黎圣母院斑马鱼设施。在这个手稿中描述的方法是通过巴黎圣母院动物护理和使用委员会批准的大学，并符合在视觉与眼科视觉研究中使用动物的研究协会的声明。 1.解决方案制备70％的乙醇消毒的组织培养罩和任何设备/被转移到组织培养罩试剂。 添加2mL的2-苯氧基乙醇的1升系统水（1：500 2-苯氧基乙醇）。 </…
Representative Results
根据图1中的示意性概述的过程的视网膜的隔离允许扁平背侧视网膜培养从光受损成人的Tg [GFAP：nGFP]在5％CO历时至少24小时的mi2004斑马鱼2 /空气环境。这些平面安装视网膜植可用于在深部组织的层次图像焦平面。一个例子是米勒胶质/标记与来自穆勒特定胶质启动子GFAP（胶质纤维酸性蛋白）的GFP神经元祖细胞的细胞核被在光?…
Discussion
研究调查执政损坏的成年斑马鱼视网膜主要用于免疫组化方法5，25，26，27，28，29，30的再生机制。建立培养视网膜外植体的条件和对现象，如INM进行活细胞成像，为我们提供了一种技术，获得深入的空间和时间信息。这种…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们赞赏威廉·阿彻和巴黎圣母院综合成像设备提供的支持。特别感谢被定向到Freimann生命科学技术人员为他们的持续帮助和他们的照顾和斑马鱼的养殖。这项研究是由美国国立卫生研究院，从圣母大学的国立眼科研究所DRH（R01-EY018417，R01-EY024519）和中心斑马鱼研究，巴黎圣母院，赠款支持。
Materials
| Dumont forceps size #5 | World precision instruments | 14098 | |
| Dumont forceps size #5, 45° angle | World precision instruments | 14101 | |
| McPherson-Vannas scissors | World precision instruments | 501233 | |
| Fluordishes | World precision instruments | FD35-100 | |
| Stereomicroscope | Nikon | SMZ-1B | similar type of dissection stereomicroscope will work |
| Biological Safety Cabinet class type A2 | Labconco | equivalent type will work | |
| tissue culture incubator | Thermoscientific | HEPA-class 100 | equivalent type will work |
| Sylvania fluorescent lamps OSFP5835HOECO | Bulbtronics | 31850 | |
| 0.2 µm pore-size Acrodisc syringe filter | VWR | 4192 | |
| 10 ml Luer-lok syringe | VWR | BD309604 | |
| 60 ml Luer-lok syringe | VWR | BD309653 | |
| NaHCO3 | FischerScientific | S233-500 | |
| CaCl2 | ThermoScientific | C79-500 | |
| MgCl2 | EMD Millipore | 5980 | |
| HBSS w/o Ca2+/Mg2+, w/o phenol red, Gibco | ThermoScientific | 14175-095 | |
| MEM w/o phenol red, Gibco | ThermoScientific | 5100-038 | |
| Horse serum, heat-inactivated | ThermoScientific | 26050-070 | |
| penicillin/streptomycin | VWR | 16777-164 | |
| Ultrapure low melting point agarose | ThermoScientific | 16520-100 | |
| ethanol, absolute | ThermoScientific | BP2818-4 | |
| 2-phenoxyethanol | Sigma | 77699 | |
| Corning Cell-Tak cell and tissue adhesive | VWR | 354240 | |
| refractive index liquid | Cargille Lab | 1803Y | |
| Nikon A1 multiphoton microscope equipped with a MaiTai infrared laser | Nikon | equivalent system will work | |
| 40x Apo long-distance water immersion objective (N.A. 1.15) | |||
| environmental chamber equipped with insert for 35 mm petridishes | Okolab | equivalent system will work | |
| NIS analysis software | Nikon |
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Cite This Article
Lahne, M., Gorsuch, R. A., Nelson, C. M., Hyde, D. R. Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy. J. Vis. Exp. (120), e55335, doi:10.3791/55335 (2017).