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, 以研究其他生物过程, 例如, 破坏赫的膜诱导脉络膜新生血管。有最小的附带损害和再生反应的时间可以精确调整和性。
随着时间的推移, 我们使用海德堡 Spectralis 系统和海德堡眼探险软件型的变化。因此, 在斑马鱼中发现眼底红外显像是特别有用的。与眼底荧光 (AF) 不同的是, 激光光斑与周围区域的对比度较低, 通过红外成像可以更容易地定位受损部位。这使我们能够检测更改并监视在体内的再生, 这在 AF 模式下是不可能的。如前所述25, OCT 可用于监视斑马鱼视网膜的退行性/再生过程。在我们的模型中, 视网膜激光损伤被检测为一个超反射带 (图 3), 这与哺乳动物在26中报告的类似。在再生过程中, 超反射信号下降, 直到14天完全消失。
OCT 图像与在激光病变中看到的形态学变化 (图 4) 相关。OCT 图像中弥漫的超反射信号 (0 天) 代表了斑马鱼在激光治疗后1小时内 INL 的 post-laser 变化。在3天检测到的微妙和界定超反射信号对应于空腔的形成, 由于杆的损耗。从14天开始, OCT 和组织学分析证实, 外视网膜已恢复其正常形态。
在本研究中, 我们也评估了 MC 反应在视网膜再生 (图 5)。许多团体已经研究了胶质细胞的激活, 特别是对 MCs 的活化, 作为对损伤的初始再生反应。更具体地说, 他们建议, 病理生理学 MC 激活可能诱导 MCs 采取干细胞的特点, 提供一个内源性的新功能细胞类型, 可以集成到受损的地区的视网膜27。正如预期的, 我们发现, 视网膜激光损伤刺激 MC 活化, 表明上调的胶质蛋白。实际上, 在后3天内, 明显增加了胶质蛋白的表达, 并仅限于损伤区。从14天开始, 再生很大程度上是完整的, 胶质细胞的信号被 downregulated 到损伤区域的基线水平。因此, 我们已经证明, MCs 发挥积极作用的视网膜重组, 并可能再生, 继受伤。
总之, 激光诱导的视网膜变性/再生模型是一种多用途的工具, 以产生快速和病灶损伤的斑马鱼视网膜和以下再生可以可视化的无侵入 OCT 成像。此外, 我们可以证明, 激光视网膜变性是伴随着 MC 活化, 随后的胶质是逆转后, 再生。然而, 需要对所涉及的细胞类型 (例如,小胶质, 巨噬) 和通路进行详细的研究。可能需要进行关键的修改, 特别是对 MCs 的实时跟踪, 以便更好地了解它们在再生过程中的行为 (例如,从它们在 INL 中的原始位置迁移到外部视网膜, 特别是在减贫战略层中)和它们对 PR 细胞的分化。另外, 光损伤范例可以被用来引起广泛和一致的损失的杆和锥型减贫战略28。然而, 所描述的模型的优势是一个更明确的伤害区域, 其中一个可以研究退化光和活化 MCs 之间的局部相互作用。
总的来说, 我们相信该模型将有助于更好地理解在感官视网膜的退行性/再生过程, 并可能使这些发展与哺乳动物系统的比较。它也可以用来研究先天免疫系统的影响和活性物质的影响。在将来, 人们可以利用这些结果来修改人类视觉系统的退化。

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&#38;L Alexa Fluor&#174; 488</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG H&#38;L Alexa Fluor&#174; 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 &#38; 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)。它从视网膜色素上皮 (RPE) 延伸到外状层 (组织)。1天也可探测到类似的信号。3天后, 这个弥漫的信号变得更有条理和密集。它在外层核层中一直可见, 延伸到感光层。在第一周 (7 天) 之后, 平均病灶大小明显下降, 只有一个小的超反射信号被检测到。从14天开始直到最新的时间点被调查 (星期 6), 激光斑点在 IR 和 OCT 图像不再是可看见的。
视网膜变性/再生的组织学评估: 为了研究视网膜变性/再生的程度和动力学, H 和 #38; E 染色在损伤诱导后的不同时间点被使用 (天 0, 1, 3, 7, 14和6周) (图 4)。对三只三只鱼的眼睛进行了实验。然而, 没有进行统计分析, 因为这份手稿的目的是演示方法。在激光治疗后60分钟内, 内部核层 (INL) 和 post-laser 的细微变化可以立即被看到 (例如,轻度水肿和 INL 细胞间隙减少)。在激光损伤诱导后6周内变性发生。1天后, 随着减贫战略的瓦解和在视网膜空间中的空洞形成, 形态学改变持续观察。事实上, 在1和7天之间的损伤区内, 核中的细胞核丢失了。最大 PR 损失在3天被发现了。从14天到6周, 外部视网膜重建了它的正常形态。
视网膜变性/再生过程中的神经胶质受累: 为了评估在不同时间点 (0、3、14) 的 MC 激活, 我们对胶质细胞标记进行了免疫组化分析, 并胶质蛋白 (图 5)。对三只三只鱼的眼睛进行了实验, 没有进行定量分析。GS 在控制胞外谷氨酸水平方面起着重要的作用, 并被认为是在 MC soma 中完全表达的, 其过程19,20。在衰老和视网膜受损或有压力时, 胶质蛋白上调。它在 MC 端脚中本地化, 并处理21。弱的胶质蛋白表达也发现在未受伤控制的视网膜标签其他胶质细胞类型,例如,星形胶质。PR 外段的自发荧光也提供了一个信号, 但这可以清楚地区分。上调3天后。因此, 在激光治疗后3天进行的分析显示, 只有在视网膜的损伤部位内才会出现胶质纤维阳性的 MCs。从14天, 再生基本上是完整的, 并 downregulated 的胶质细胞信号的基线水平的损害区域。与胶质蛋白不同, GS 表达水平没有显著变化。然而, 在受伤地点可能有局部下调的 GS。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56249/56249fig1.jpg"> 
图 1: 对斑马鱼视网膜激光损伤的诱导和 OCT 分析的设置.(A) 在治疗前安排激光系统。(1) 在应用激光束之前, 将斑马鱼置于硅胶针架上, 并放置在眼底接触镜上。(B) 在影像学前安排 OCT 系统。(1) 斑马鱼放置前78D 狭缝灯镜头前, 通过 OCT 检测损伤部位.<a href="//ecsource.jove.com/files/ftp_upload/56249/56249fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56249/56249fig2.jpg"> 
图 2: 用于获取 IR 和 OCT 图像的设置.(A) 图像中软件窗口的屏幕截图。描述了 IR 和 OCT 模式。(B) 用于获取图像的小组概览。<a href="//ecsource.jove.com/files/ftp_upload/56249/56249fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56249/56249fig3.jpg"> 
图 3: 激光诱导的视网膜变性后, 在不同时间点 (0、1、3、7、14和6周) 同一眼内视网膜损伤和未受伤部位的 IR (左) 和 OCT (右) 图像.在红外图像中, 绿色方框显示了已被分析的视网膜部分, 绿色的线表明了 OCT 图像在视网膜上的位置。箭头表示在 OCT 中检测到的激光斑点的位置为超反射信号. 缩放栏 = 200 µm.<a href="//ecsource.jove.com/files/ftp_upload/56249/56249fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56249/56249fig4.jpg"> 
图 4: H 和 #38; 在不同时间点 (0、1、3、7、14和6周), 在激光诱导的视网膜变性后, 损伤和对侧未受伤的斑马鱼眼的染色.气相色谱, 节细胞层;INL, 内核层;只有外核层缩放栏 = 50 µm.<a href="//ecsource.jove.com/files/ftp_upload/56249/56249fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56249/56249fig5.jpg"> 
图 5: MC 激活的免疫组织化学检测在不同时间点 (0、3、14) 的受损伤和未受伤的斑马鱼视网膜上的胶质纤维 (绿色) 和 GS (红色) 染色, 导致视网膜变性。在唯一的绿色信号是由于荧光的公关 o外段。细胞核被 DAPI (蓝色) 染色。缩放栏 = 50 µm.<a href="//ecsource.jove.com/files/ftp_upload/56249/56249fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about MÃƒÂ¼ller 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

Nanyang Technological University

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Optogenetic激活斑马鱼的体感神经元使用厨师tdTomato的

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

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, ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.