Name: müller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish
Uploaded: 2017-10-27T14:00:00.000+00:00
Duration: 6 min 27 s
Description: Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

我们感谢马丁 Zinkernagel, MD, 博士和仪 Reisenhofer, 博士为她的科学投入建立模型和费德里卡比西纳尼为她出色的技术援助。

所有实验都遵守了在眼科和视力研究协会 (帕特) 视觉研究中使用动物的声明, 并尊重政府当局的相关规定. 
 1. 动物 <ol> <li> 保持 TgBAC (胶质蛋白: GFP) 斑马鱼 167 (AB) 菌株在水中的标准条件下 6-9 月, 温度为26.5 和 #176; C 和 14/10 h 光/暗循环 18 . </li>
	<li> 经政府当局批准后, 按照有关机构的动物保育准则进行动物实验. </li>
</ol> 2。可逆全身麻醉 <ol> <li> 准备 3-氨基磺酸乙酯盐 (tricaine) 的库存溶液, 溶解400毫克的 tricaine 粉, 在97.9 毫升的罐水和2.1 毫升的三缓冲盐水 (TBS) 的1米。调整到 ph 值7.0 与 1 M 三 (ph 9) 和存储在4和 #176; C 在黑暗中长达一个月. 
	注: Tricaine 应在水中准备, 如动物的自然生活条件, 最好使用原池水. </li>
	<li> 在油箱水中稀释库存溶液 1:25, 并立即使用. </li>
	<li> 将斑马鱼放入10厘米的培养皿中, 其中含有50毫升的麻醉液, 直到它们变得不动并且不响应外部刺激 (大约 2-5 分钟, 取决于重量和年龄). </li>
	<li> 将每条鱼手工传送到 custom-made 硅胶针架上进行激光处理 ( 图 1A ). 
	谨慎!鱼可以保持麻醉外的坦克, 只有10分钟. </li>
	<li> 在处理和/或成像后进行反向麻醉, 将斑马鱼放在装有水箱水的容器中. </li>
	<li> 为了支持恢复, 通过在水中来回移动斑马鱼, 在鳃上形成新鲜的水箱水. </li>
</ol> 3。视网膜激光局灶性损伤 注: 532 nm 二极管激光器用于在斑马鱼的视网膜上制造焦光损伤。这种激光的实验装置能够在成年斑马鱼中建立一个可再生的局灶性视网膜损伤. 
<ol> <li> 设置激光器的输出功率:70 mW; 天线直径:50 和 #181; m; 脉冲持续时间: 100 毫秒 警告!使用激光光源需要对该区域进行适当的个人保护和标签. </li>
	在治疗前, <li> 在眼部应用 1-2 滴 2% hydroxypropylmethylcellulose, 并使用2.0 毫米眼底激光透镜将激光瞄准光束聚焦在视网膜上. 
	谨慎!hydroxypropylmethylcellulose 滴是粘性的, 如果它在鳃上, 可能会引起呼吸问题. </li>
	<li> 在左眼周围放置四激光光斑, 并使用右、未经处理的眼睛作为内部控制. </li>
</ol> 4。 在体内 视网膜形态学的影像学 <ol> <li> 0 天, 直接在激光诱导后将斑马鱼视网膜可视化, 而不使其从麻醉中恢复。在所有其他时间点, 使用全身麻醉 (见2节: 可逆全身麻醉)。将固定的斑马鱼放在 custom-made 硅胶针架上 ( 图 1 b, b. 1 )。
	<li> 要获得最佳图像, 请切割一个商业可用的水凝胶隐形眼镜, 以适合斑马鱼眼 (和 #216; = 5.2 毫米, r = 2.70 mm, 中心厚度 = 0.4 mm), 方法是通过打孔。用纤维素填充镜片的凹面, 并将其置于角膜上. </li>
	<li> 为 OCT 系统配备78D 非接触式狭缝灯透镜. </li>
	<li> 将红外线 (ir) 图像聚焦在 ir + OCT 模式 ( 图 2A ) 中, 通过单击和 #34 来可视化眼睛的眼底, 并获取 ir 图片; 获得和 #34; 按钮 ( 图 2B ) 以本地化使用系统和 #39 软件的视网膜上的激光斑点. </li>
	<li> 在 IR + OCT 模式下可视化视网膜图层的三维剖面, 并通过单击 #34、获取和 #34; 按钮 ( 图 2B ) 来获取图片。观察外核层损伤的严重性 (参见3节: 视网膜激光局灶损伤) 在这些图像中. </li>
	<li> 在处理和/或成像后将斑马鱼放在装有水箱水的容器中, 进行反向麻醉. </li>
	<li> 为了支持恢复, 通过在水中来回移动斑马鱼, 在鳃上形成新鲜的水箱水. </li>
	<li> 在1、3、7、14和6周, 在活体 视网膜形态学成像中执行类似 . </li>
</ol> 5。苏木素和 #38; 曙红 (H 和 #38; E) 染色 <ol> <li> 安乐斑马鱼被浸没到寒冷 (4 和 #176; C) 麻醉溶液在冰上至少10分钟, enucleate 眼睛立即通过小弯曲钳. </li>
	<li> 将4% 甲醛 (粉煤灰) 的全眼固定在4和 #176 磷酸盐缓冲盐水 (PBS) 中; C 为20小时, 然后脱水的样品在一个分级酒精系列 (二甲苯100% 为5分钟两次, 乙醇100% 为5分钟两次, 乙醇96% 为3分钟两次, 乙醇 70% 3 min 次). 
	谨慎!粉煤灰可以刺激眼睛, 鼻子和上呼吸道的轨道。粉煤灰是一种已知的人类致癌物和疑似生殖危险. </li>
	<li> 将样品嵌入石蜡切片, 切割5和 #181; m 部分在视神经头的水平, 并安装在玻璃幻灯片上. </li>
	<li> 用0.1% 酸苏木精溶液将 deparaffinized 切片染色5分钟, 然后在蒸馏水中蘸上两次在盐酸混合 (2 毫升 25% HCl 中250毫升蒸馏水) 和氨混合 (2 毫升25% 的氨水) 中的幻灯片。蒸馏水)。染色的部分与曙红 G 水溶液0.5% 为3分钟后, 发展的苏木精染色自来水至少10分钟. </li>
	<li> 在丙烯酸树脂安装介质上安装脱水的幻灯片, 并在光显微镜下观察幻灯片. </li>
</ol> 6。MC 激活的免疫组织化学作用 <ol> <li> 将抗原检索缓冲区中的 deparaffinized 部分加热 (三-EDTA + 0.05% 非离子洗涤剂, pH 9.0) 3 分钟在适当的蒸笼或微波炉 10-15 分钟和洗涤三次与 TBS 为5每分钟. </li>
	<li> 用硅胶笔圈出部分, 并添加100和 #181; L 阻断溶液 (TBS + 10% 山羊正常血清 + 1% 牛血清白蛋白, pH 7.6) 在室温下为 1 h. </li>
	<li> 染色与 anti-glial 纤维酸性蛋白 (胶质酸) 多克隆抗体和 anti-glutamine 合成酶 (GS) 小鼠单克隆抗体, 都在1:200 稀释 (40 和 #181; L 每样)。在4和 #176 ° C 的湿室中孵育幻灯片。用 TBS + 0.1% 吐温20洗三次, 每次5分钟. </li>
	<li> 用适当的辅助抗体完成可视化。该协议使用山羊兔 igg h 和 #38; 绿色二次抗体和山羊 anti-mouse igg 和 #38; l 亮红色的 GS, 在1:500 稀释在室温下为1小时. </li>
	<li> 装入包含 DAPI 的安装介质的幻灯片, 并在荧光显微镜下观察幻灯片. </li>
</ol>

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作者没有什么可透露的。

通过毒素介导的细胞死亡22、机械损伤23和热损伤24等不同方法对斑马鱼视网膜再生/变性进行了研究。我们使用了 532 nm 二极管激光器来破坏斑马鱼视网膜。因此, 我们的模型提供了几个优点。例如, 我们迅速建立了一个明确界定的损伤区域, 在外部视网膜, 特别是在减贫战略层。此外, 这种实验装置可以被修改, 以产生较大的损害区域, 以研究其?...

视力可能是人类最基本的感觉, 它的损害具有很高的社会经济影响。在工业化世界中, 视网膜退行性疾病占成年人群视力丧失和失明的大多数1。视网膜色素变性 (RP) 是最常见的遗传性失明的原因, 在人的年龄之间的20和 60, 影响约150万人在全球范围内2,3。这是一个异构家族遗传性视网膜疾病的特点是逐步丧失的光 (减贫) 后, 视网膜色素上皮变性, 随后, 胶质和重塑内神经元4。疾病的过程可以解释的两个 PR 细胞类型的增量损失, 通常从棒开始, 这是负责的消色差视觉在昏暗的光和锥, 这是必不可少的颜色视觉和视力5。单个基因缺陷足以引起 RP。到目前为止, 超过45基因的130突变已经与疾病6相关。这导致不同的疾病表型, 是一个原因, 基因治疗是 non-generalizable, 从而一个复杂的治疗方法。因此, 迫切需要制定新的一般治疗方法来治疗致盲疾病的视网膜退化。
视网膜变性常涉及 PR 损失;因此, PR 细胞死亡是视网膜退行性过程的标志7。它已经证明, PR 细胞死亡刺激 m ü ller 胶质细胞 (MC) 激活和增殖8。MCs, 在脊椎动物视网膜的主要胶质细胞类型, 曾经被认为只是一个 "胶水" 之间的视网膜神经元。近年来, 许多研究表明, MCs 的作用不仅仅是结构支持9。在不同的功能中, MCs 也参与神经再生和修复10。事实上, 在对变性视网膜的扩散因素的反应中, MCs 显著增加胶质纤维酸性蛋白的表达。因此, 在视网膜损伤和变性11中, 胶质蛋白标签可以作为 MC 活化的标志。
最近, 我们开发了一种新的局灶损伤的适应, 利用激光诱导斑马鱼视网膜变性 (斑马鱼)。局灶性损伤有利于研究某些生物学过程, 如细胞迁移到受伤部位, 以及视网膜再生过程中发生的事件的准确时间12。此外, 由于其视觉系统与其他脊椎动物的相似性, 斑马鱼在视觉研究中已经变得非常重要。人和硬视网膜的大体形态和组织学特征显示出很少的差异。因此, 人类和斑马鱼视网膜包含相同的细胞类组织在同一层模式, 其中感光光占据最外层, 而视网膜投射神经元, 神经节细胞, 居住在最里面神经元层, 近于晶状体。视网膜神经元, 突, 两极, 和水平细胞, 定位在感光细胞和神经节单元层之间13。此外, 斑马鱼视网膜是锥体控制, 因此更接近人的视网膜比, 例如, 密集研究的啮齿动物视网膜。硬和哺乳动物之间的一个奇妙的区别是鱼类视网膜的持续神经再生和损伤后的视网膜重建。在斑马鱼, MCs 能分化和斡旋再生在受伤的视网膜14,15。在鸡肉中, MCs 也有能力重新进入细胞周期和分化。在成年鱼类视网膜损伤后, MCs 采用了祖细胞和干细胞的某些特征, 迁移到受损的视网膜组织, 产生新的神经元16。哺乳动物 mcs 的基因表达谱显示出与视网膜祖细胞有意想不到的相似性, 而在鸡、啮齿动物甚至人类视网膜中, mcs 的内在神经源潜能的证据也在增长17。然而, 为什么鸟类和哺乳动物的再生反应比起鱼类的健壮反应还要低呢？因此, 了解斑马鱼内源性修复机制可能会提出刺激哺乳动物和人类视网膜再生的策略。采用 MCs 内源性修复机制作为治疗视网膜变性患者的治疗工具, 将对我们的社会产生显著的影响。
在此, 我们提供了在眼科研究中使用变性/再生模型的必要步骤。我们首先关注的是诱导感觉神经视网膜的病灶损伤, 然后是在损伤部位发生的事件的成像, 最后是对相邻 MCs 的参与。一般的协议是相对容易执行, 并打开了各种可能性, 评估视网膜后。

<table><tbody><tr><td>Acid hematoxylin solution</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>2852</td><td></td></tr><tr><td>Albumin</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>A07030</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>5470</td><td></td></tr><tr><td>Dako Pen</td><td>Dako, Glostrup, Danmark</td><td>S2002</td><td></td></tr><tr><td>DAPI mounting medium</td><td>Vector Labs, Burlingame, CA, USA</td><td>H-1200</td><td></td></tr><tr><td>Eosin G aqueous solution 0.5%</td><td>Carl Roth, Arlesheim, Switzerland</td><td>X883.2</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>2860</td><td></td></tr><tr><td>Ethylene diamine tetraacetic acid (EDTA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>ED</td><td></td></tr><tr><td>Eukitt</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>3989</td><td></td></tr><tr><td>Goat anti-rabbit IgG H&amp;amp;L Alexa Fluor&amp;reg; 488</td><td>Life Technologies, Zug, Switzerland</td><td>A11008</td><td></td></tr><tr><td>Goat anti-mouse IgG H&amp;amp;L Alexa Fluor&amp;reg; 594</td><td>Life Technologies, Zug, Switzerland</td><td>A11020</td><td></td></tr><tr><td>Goat normal serum</td><td>Dako, Glostrup, Danmark</td><td>X0907</td><td></td></tr><tr><td>Hydrogel contact lens</td><td>Johnson &amp;amp; Johnson AG, Zug, Switzerland</td><td>n.a.</td><td>1-Day Acuvue Moist</td></tr><tr><td>Hydroxypropylmethylcellulose 2%</td><td>OmniVision, Neuhausen, Switzerland</td><td>n.a.</td><td>Methocel 2%</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>A5040</td><td>Tricaine, MS-222</td></tr><tr><td>Visulas 532s</td><td>Carl Zeiss Meditec AG, Oberkochen, Germany</td><td>n.a.</td><td>532 nm laser</td></tr><tr><td>Mouse anti-GS monoclonal antibody</td><td>Millipore, Billerica, MA, USA</td><td>MAB302</td><td></td></tr><tr><td>HRA + OCT Imaging System</td><td>Heidelberg Engineering, Heidelberg, Germany</td><td>n.a.</td><td>Spectralis</td></tr><tr><td>Heidelberg Eye Explorer</td><td>Heidelberg Engineering, Heidelberg, Germany</td><td>n.a.</td><td>Version 1.9.10.0</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5368</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5368</td><td></td></tr><tr><td>Rabbit anti-GFAP polyclonal antibody</td><td>Invitrogen, Waltham, MA, USA</td><td>180063</td><td></td></tr><tr><td>Silicone pin holder</td><td>Huco Vision AG Switzerland</td><td>n.a.</td><td>Cut by hand from silicone pin mat of the sterilization tray accordingly.</td></tr><tr><td>Slit lamp BM900</td><td>Haag-Streit AG, Koeniz, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>Slit lamp adapter</td><td>Iridex Corp., Mountain View, CA, USA</td><td>n.a.</td><td></td></tr><tr><td>Superfrost Plus glass slides</td><td>Gehard Menzel GmbH, Braunschweig, Germany</td><td>10149870</td><td></td></tr><tr><td>TgBAC (gfap:gfap-GFP) zf167 (AB) strain</td><td>KIT, Karlsruhe, Germany</td><td>15204</td><td>http://zfin.org/ZDB-ALT-100308-3</td></tr><tr><td>Tris buffered saline (TBS)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5912</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P1379</td><td></td></tr><tr><td>78D non-contact slit lamp lens</td><td>Volk Optical, Mentor, OH, USA</td><td>V78C</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>534056</td><td></td></tr><tr><td>Ocular fundus laser lens</td><td>Ocular Instruments, Bellevue, WA, USA</td><td>OFA2-0</td><td></td></tr><tr><td>2100 Retriever</td><td>Aptum Biologics Ltd., Southampton, United Kingdom</td><td>R2100-EU</td><td>Steamer</td></tr></tbody></table>

m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish

硬和哺乳动物之间的一个奇妙的区别是硬视网膜在严重损伤后的视网膜神经形成和再生的终身潜能。调查斑马鱼的再生途径可能带来新的见解, 以制定创新的策略, 治疗视网膜退行性疾病的哺乳动物。在此, 我们的重点是通过 532 nm 半导体激光器诱导成人斑马鱼外侧视网膜病灶。局部损伤允许对视网膜变性和在损伤区域直接再生的生物过程进行调查。使用无创光学相干断层扫描 (OCT), 我们能够定义受损区域的位置, 并监测后续再生在体内.事实上, OCT 成像产生的高分辨率, 横断面图像的斑马鱼视网膜, 提供的信息, 以前只提供了组织学分析。为了证实 real-time OCT 的数据, 通过免疫组织化学研究视网膜损伤诱导后的组织学切片和再生反应。

实时 OCT: 为了分析 MCs 在视网膜修复中的作用, 我们使用了一种激光损伤模型, 在斑马鱼视网膜中诱发界定损伤区。损伤部位是在受伤后的60分钟内第一次 (0 天) 用在体内OCT 成像的 (图 3)。为了补偿鱼眼的光学, 在角膜上放置了一个 custom-made 的隐形眼镜。激光治疗后, 一个弥漫性超反射信号被定位到外部视网膜 (箭头)。它从视网膜色?...

Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

斑马鱼是研究脊椎动物视网膜变性/再生机制的流行动物模型。本议定书描述一种方法, 以诱导局部损伤破坏外视网膜, 对内视网膜的损害最小。随后, 我们监测在体内视网膜形态学和 m ü ller 胶质细胞反应在整个视网膜再生。

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

mller-glia-cell-activation-laser-induced-retinal-degeneration

Research

JoVE Journal

Neuroscience

9.7K 次观看.  University of Bern. 斑马鱼是研究脊椎动物视网膜变性/再生机制的流行动物模型。本议定书描述一种方法, 以诱导局部损伤破坏外视网膜, 对内视网膜的损害最小。随后, 我们监测在体内视网膜形态学和 m ü ller 胶质细胞反应在整个视网膜再生。

视频: Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

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

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

科研

发育生物学

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

神经科学

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

生物学

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

原代细胞培养物研究小鼠穆勒胶质细胞在微RNA处理后的再生潜力

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.
A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.
In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

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

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...