Methylnitrosourea（MNU）诱导的视网膜变性和再生的斑马鱼：组织学和功能特征
Summary
在这里我们展示视网膜脱离和再生，对以N甲基 -N- -nitrosourea在成年斑马鱼的视觉功能的影响进行量化。其次是在内核层扩散视力丧失和降低光感受器号。发生完整的形态和功能恢复的初步治疗后30天。
Abstract
视网膜退行性疾病， 如视网膜色素变性，用得到的，为广大视力下降，在工业领域感光伤害帐户。动物模型是至关重要的意义，研究这种疾病。在这方面，在感光体特异性毒素N-甲基-N- -nitrosourea（MNU）已被广泛用于在啮齿动物药理学诱导的视网膜变性。此前，我们已经建立了MNU诱导的视网膜变性模型中的斑马鱼，在视觉研究的另一种流行的模型系统。
一个迷人的区别是哺乳动物在成年斑马鱼视网膜损伤后的再生持续神经。为了量化这一观察，我们在成年斑马鱼使用视力的测量。由此，视动反射被用于跟随在非麻醉的鱼的功能变化。这补充了组织学以及免疫staini吴凋亡（TUNEL）和增殖（PCNA）关联的发展形态变化。
综上所述，光感受器细胞凋亡MNU治疗，随后是细胞中的外核层（ONL）的显着降低三天之后出现。此后，将细胞在内核层（INL）和ONL的增殖被观察。在此，我们表明，不仅是一个完整的组织，而且功能性再生发生在30天的时间过程。现在我们说明的方法来量化和跟进的视频格式使用的MNU斑马鱼视网膜脱离和再生。
Introduction
视觉是最重要的意义的人及其损害具有较高的社会经济影响。在发达国家，视网膜变性疾病是视力减退和失明的老年人中1的主要原因。最退行性视网膜疾病的原因只是部分了解和治疗方案，以夺回失去的视力是非常有限的。视网膜色素变性是视网膜变性疾病的原发性感光损失2-3。n的典型例子甲基 -N -nitrosourea（MNU）诱导的视网膜变性，因此被广泛应用在啮齿类动物模型与主光感受器细胞死亡4疾病。它是一种烷化剂，并导致良性和恶性肿瘤，通常会出现曝光5-7后几个月。此外，它会导致短期的观察期间内特定光感受器细胞死亡。由此，视网膜层structu的损失重和显著视网膜变薄了以浓度依赖性方式观察到。视网膜神经胶质细胞被激活，但在视网膜色素上皮细胞（RPE）没有变化被发现。内质网（ER）应激相关的细胞凋亡似乎是MNU作用的主要途径在视网膜8。
我们最近推出的MNU作为一种化学模型诱导光感受器变性斑马鱼9。除了 其他原因，斑马鱼（ 斑马鱼 ）已成为因为它的视觉系统对其他脊椎动物10的相似之处在视觉研究很重要。外层视网膜包含感光体，它可被分成四个不同的锥面类型具有峰值灵敏度的紫外线，短，中等，和可见光谱的长波长和一个视杆型。在内核层（INL）的双极性，水平和无长突的interneurons细胞体被发现，如WELl与穆勒胶质细胞的胞体。在所述外丛状层（OPL）的光感受器和视网膜内层之间的突触联系形成的，而最接近透镜的细胞层是神经节细胞层（GC），该组件形成长轴突包括视神经和视束。神经节细胞和细胞中的内核层之间的突触联系形成于内网层（IPL）11。视网膜色素上皮所在的视网膜神经上皮层外，并围绕光感受器外段长微绒毛顶端12。此外，斑马鱼是高度再生，并能够再生毁损脑，视网膜，脊髓，心脏和其他组织13。当视网膜损伤时，细胞中的INL，这被认为是Müller细胞，被激活并具有分化成多种视网膜细胞类型的潜力。此外，它们还产生棒祖细胞，它们分别位于外核层。另一个让urce供给成年斑马鱼视网膜有新生细胞的纤毛边缘区。这个源是需要实现的视杆细胞的恒定密度的不断增长的斑马鱼眼14。
在MNU模型可以用作视网膜组织的简单和可再现的变性/再生方法。由于在斑马鱼中的生物过程的某些相似性和在人体中，这可能打开门，以确定参与细胞死亡途径，筛选潜在的神经保护药物。根据我们组先前的研究，我们现在说明这MNU诱导斑马鱼视网膜脱离和随之而来的再生包括根据实验室影片9功能变化模型的方法。
Protocol
Representative Results
Discussion
此前，本集团已转移光感受器变性的MNU模式从啮齿动物到斑马鱼系统9。随后的事件进行了描述，并随访了30天。在这段时间内完整视网膜形态退化和再生的初始治疗后发生。首先，组织学显示，从3天减少杆细胞计数与最小的相应8天，TUNEL染色MNU处理后第3天确定的视杆细胞的凋亡。再生开始的INL为3天，最长为5天外核层，增殖细胞可观察到的观察期，最大的结尾处5天，这是与此相反的INL在…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Monika Kilchenmann, Federica Bisignani and Agathe Duda for their excellent technical assistance.
Materials
| Acetic acid | Sigma-Aldrich, Buchs, Switzerland | A6283 | |
| Ammonia | Sigma-Aldrich, Buchs, Switzerland | 294993 | 0.80% |
| Bovine serum albumine (BSA) | Sigma-Aldrich, Buchs, Switzerland | 05470 | |
| Dako Pen | Dako, Glostrup, Danmark | S2002 | |
| DAPI mounting medium | Vector Labs, Burlingame, CA, USA | H-1200 | |
| Eosin | Sigma-Aldrich, Buchs, Switzerland | 45260 | |
| Ethanol | Sigma-Aldrich, Buchs, Switzerland | 2860 | 100%, 96%, 70% |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich, Buchs, Switzerland | ED | |
| Ethyl 3-aminobenzoate methanesulfonate salt | Sigma-Aldrich, Buchs, Switzerland | E10521 | Tricaine |
| Eukitt | Sigma-Aldrich, Buchs, Switzerland | 3989 | |
| Goat anti-rabbit Alexa 594 | Life Technologies, Zug, Switzerland | A11012 | |
| Goat normal serum | Dako, Glostrup, Danmark | X0907 | |
| Hydrochloric acid | Sigma-Aldrich, Buchs, Switzerland | 320331 | 0.20% |
| In situ Cell Death Detection Kit | Roche Applied Sciences, Rotkreuz, Switzerland | 11684795910 | TUNEL Kit |
| Mayer's hemalum solution | Merck, Darmstadt, Germany | 109249 | |
| Methylnitrosourea (MNU) | Sigma-Aldrich, Buchs, Switzerland | N4766 | Toxic |
| OptoMotry | CerebralMechanics, Lethbridge, AB, Canada | n.a. | |
| Paraformaldehyde (PFA) | Sigma-Aldrich, Buchs, Switzerland | P6148 | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich, Buchs, Switzerland | P5368 | |
| Proteinase K | Dako, Glostrup, Danmark | S3004 | |
| Rabbit anti-PCNA | Santa Cruz Biotechnology, Santa Cruz, USA | sc-33756 | |
| Superfrost Plus glass slides | Gehard Menzel GmbH, Braunschweig, Germany | 10149870 | |
| Tris buffered saline (TBS) | Sigma-Aldrich, Buchs, Switzerland | P5912 | |
| TrizmaÒ base | Sigma-Aldrich, Buchs, Switzerland | T1503 | |
| Tween 20 | Sigma-Aldrich, Buchs, Switzerland | P1379 | |
| Xylene | Sigma-Aldrich, Buchs, Switzerland | 534056 | |
| Zebrafish (Danio rerio) AB (Oregon) strain | University of Fribourg, Dept. of Biology | n.a. | Own fish facility |
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