We thank N. Borel and her fish house team, M. Rastegar for help with microscopy, and T. Dickmeis for comments on the manuscript. We are grateful for support by the EU IP ZF-Health, FP7-HEALTH-2007-B2, NeuroXsys, the Interreg Network for Synthetic Biology in the Upper Rhine valley (NSB-Upper Rhine) and the BMBF funded network EraSysBio.

斑马鱼保持在毒物学和遗传学（ITG）的研究所科技（KIT）的卡尔斯鲁厄理工学院的鱼设施。动物实验是在按照德国动物保护的标准进行的，并经政府批准巴登 - 符腾堡，Regierungspräsidium德国卡尔斯鲁厄（Aktenzeichen 35-9185.81/G-272/12&#39;Adulte Neurogenese&#39;）。 1，生成刀伤在端脑<ol><li>麻醉<ol><li>以制备三卡因原液（3 - 氨基苯甲酸乙酯，也称为乙基-3 - 氨基苯甲酸酯）使用400毫克三卡因粉末，溶于97.9毫升水和2.1毫升的1摩尔的Tris / HCl缓冲液（pH为9）。将pH调节至7。使每份5毫升，并将其储存在-20℃直到使用。 </li><li>转让0.5-1岁斑马鱼的适当数量从他们的坦克进入塑料鼠笼。注:成年斑马鱼在大约12头骨月龄还比较软，可以用针容易穿孔（参见第1.2.3节和图1A）。 </li><li>麻醉的成年斑马鱼，结合5毫升三卡因（原液）在一个塑料交叉笼约100毫升干净的鱼缸水（三卡因终浓度为0.02％（W ​​/ V）三卡因）。 </li><li>孵化鱼在麻药，直到他们不动了（45-60秒）。 </li></ol></li><li>端脑损伤<ol><li>个别地方麻醉斑马鱼成的三卡因浸泡过的泡沫塑料块的缝隙。 </li><li>在解剖显微镜下用光线从顶部用一只手轻轻握住鱼和它定位的方式，允许从顶部进入头部。 </li><li>用另一只手垂直穿过颅骨成一个端脑半球（ 图1A-1B）的内侧区按一个30 G注射器（胰岛素注射器带有集成针）。端脑的一个半球通过头骨重新可见。确保所引入的病变是不大于2毫米（ 图1C）更深。注:强烈建议总是引进病变到同一个半球解剖出脑颅骨时，缓解病变部位的鉴定。 </li><li>引入端脑损伤后，将鱼放入新鲜的鱼水。保持多达10个鱼在2升鼠笼充满鱼水进行回收和鼠笼连接到水流动系统。注:成活率一般为〜97％，如果鱼是健康的，没有其他的实验用鱼之前执行。除抗生素对鱼的水是没有必要的。恢复后，鱼通常的行为:他们游泳，饲料，和队友。 </li></ol></li></ol> 2，分析端脑损伤的影响<ol><li>大脑解剖和固定<ol><li>在不同时间点后病变（。典型地重新麻醉恢复的鱼Y，1，3，5和14天后病变（DPL））与0.02％（重量/体积）三卡因如上和通过将它们放入冰水中5分钟安乐死它们。 </li><li>将鱼放在纸巾浸泡在磷酸盐缓冲盐水（PBS）中，并通过切割鳃后面用锋利的剪刀从主体分离的头部。 </li><li>保持磁头在1x PBS中5分钟，以便使止血。注:这样可以释放血液从组织，这可能扰乱在协议采取进一步措施。 </li><li>过夜固定，在4℃下在4％多聚甲醛（PFA）的PBS中，或4小时，在室温（RT）的磁头。 </li><li>在培养皿用1×PBS洗涤两次固定磁头。 </li><li>在解剖显微镜下，23在1x PBS中仔细解剖大脑。病灶通常在解剖显微镜下可见。大脑没有明显的病变应该被丢弃。 </li><li>转移大脑分为2毫升的反应管中填充用1.5ml 100％甲醇（甲醇）和反转管5倍孵化前它们在-20℃下至少16小时。注意:大脑可以保持几个月的MeOH在-20℃，直到需要进行免疫组化或原位杂交。 </li></ol></li><li>组织制备的免疫组化<ol><li>通过递减甲醇系列在2ml塑料管（依次75％甲醇（1×PBS，0.1％吐温20 = PTW）再水合的大脑; 50％甲醇，25％MeOH中孵育大脑（大脑15/2毫升的反应管）5分钟，在各溶液中。 </li><li>在PTW 5分钟洗脑筋。用新鲜的PTW重复4倍。 </li></ol></li><li>嵌入大脑的切片<ol><li>嵌入，从2毫升的反应管中的PTW用​​移液管（直径5mm）传输到大脑的聚乙烯成型杯托盘的空腔。 </li><li>制备2％琼脂糖（DNA级）中的1×PBS中的锥瓶中。加热它在微波中在600 W直至琼脂糖完全溶解。制备50毫升10绞尽脑汁。让日Ë琼脂糖冷却3分钟，在室温下使用前。保持预热的加热块（60℃），而嵌入的大脑中，为了防止凝固的琼脂糖。 </li><li>通过使用巴氏吸管（1毫米直径）从含有​​一种脑模具中取出的1×PBS。小心不要损伤大脑。 </li><li>倒入琼脂糖倒入模具，完全填充它。然后用解剖针漩涡大脑在琼脂糖洗去PTW使其更容易嵌入大脑。 </li><li>东方大脑在模具腹朝下，背部朝上，并把它笔直。 </li><li>让琼脂糖冷却下来。为了更快地冷却，放在冰模。重复步骤。 </li><li>所有的大脑重复2.3.3-2.3.6。 </li></ol></li><li>修剪琼脂糖块<ol><li>用解剖针，蹦出琼脂糖块。 </li><li>用锋利的剃刀刀片，切琼脂糖在大脑的后端。请确保该平面平行于德lencephalon。 </li><li>切琼脂糖在大脑的前端。然后翻转块，使其看台上的后平面。 </li><li>通过削减在平行于大脑的背，腹两侧去除多余的琼脂糖。然后翻转脑回，使它在于它的腹侧。 </li><li>切割块到一个截头三角形，其中，端脑位于较小的平面（ 图2）。 </li></ol></li><li>准备Vibratome的切片<ol><li>根据制造商的说明准备vibratome。使用新的刀片和后10绞尽脑汁更换。 </li><li>用1×PBS填充缓冲区浴中，仅仅达到叶片的底部的水平。 </li><li>放置一个小点的强力胶上vibratome的检体盘的顶部。 </li><li>小心地将是位于后方的大脑上的强力胶的平面。胶水将尽快硬化，因为它是与1X PBS接触在vibratome缓冲浴。 </li><li>将样品盘与进样使用切片机的机械手的缓冲盘。 </li><li>使用机械手来旋转样品盘到所需的位置。定向在缓冲液中的块中用于定位只是在表面下的端脑的方式，朝向叶片的大脑背侧部分。然后拧紧SCRW用3毫米内六角扳手，取下机械手。注意:该夹持螺钉或夹紧装置中的一个不能位于比在试样圆盘的间隙;在这些位置夹持试样盘是不可能的。 </li></ol></li><li>切片的脑<ol><li>准备一个24孔板收集部分。每个脑被剖开，填充1以及用1毫升封闭缓冲液（0.1％（V / V）10％吐温-20，0.2％（重量/体积）牛血清白蛋白（BSA），1％（体积/体积）二甲基亚砜（DMSO）的PBS中）。 </li><li>开始切片，在50微米厚，1mm /秒的速度，和70赫兹的频率用振动切片机。 </li><li>使用合成刷（1毫米）拿起薄片琼脂糖，因为它们脱落的叶片，并收集他们在24孔板。 </li></ol></li><li>免疫组化在Vibratome节<ol><li>在封闭缓冲液（0.1％（V / V）10％吐温20，0.2％（重量/体积）牛血清白蛋白，1％（体积/体积）的DMSO的PBS）阻断非特异性结合位点1小时，在室温下。 </li><li>除去上清，加入250微升的抗体稀释于封闭缓冲液中过夜，在4℃下可替换地，孵育一抗为2小时，在室温。然后在PTW洗3次10分钟。注意:对于PCNA染色用1:400稀释（鼠抗PCNA详见表材料）。多个初级抗体可以在同一时间进行孵育。 </li><li>孵育在二次抗体的部分为2小时，在室温，然后在PTW洗3次10分钟。注:1:1,000稀释PTW，详细见表格材料。多个次级抗体可以在同一时间进行孵育。 </li><li&gt;可选:当部分被保持在PTW过夜，在4℃，以允许洗出非​​特异性结合的第二抗体的最好的结果是观察到的。在这种情况下，在安装前再次更换PTW。 </li></ol></li><li> 原位杂交成人脑的RNA（或者以免疫组化） <ol><li>大脑解剖和固定（相同的步骤进行免疫组化）: <ol><li>杂交探针。 </li><li>如所述进行免疫组化在2毫升反应管再水合的大脑。 </li><li>在PTW 30分钟，在孵育蛋白酶K（10μg/ ml的终浓度ProtK。）的大脑。经过孵化时间删除ProtK / PTW并再次修复大脑20分钟的BT-修复（4％PFA，4％蔗糖，0.12毫米氯化钙2，77毫米的Na 2 HPO 4，23毫米的NaH 2 PO 4）。 </li><li> 5分钟在PTW（0.1％（体积/ Tween-20的PBS中V））洗涤大脑5倍。 </li><li>删除PTW和冲洗的大脑在500微升HYB缓冲液（50％（体积/体积）的去离子甲酰胺，5×SSC，500μg/ ml的酵母tRNA，50微克/毫升肝素，0.1％Tween-20中，9毫citricacid）。等到大脑沉到试管底部，然后弃去上清液，加入500μl的清洁HYB缓冲区和孵化在65-70℃下进行3-4小时。 </li><li>稀释的RNA探针，以1:200或1:400（取决于探头）中的HYB缓冲400μl的最终体积。加热该稀释的RNA探针在70℃下5分钟。 </li><li>从大脑取出上清液，加入RNA探针，孵育65-70°C（O / N）。所有的洗涤缓冲液应预热并保持在70℃。 </li></ol></li><li>标签<ol><li>在65-70℃（在所有4个洗涤步骤）用500μl的洗涤缓冲液1（50％甲酰胺，1×SSC，0.05％吐温20）洗涤两次30分钟洗涤的大脑。更换缓冲液1通过缓冲器2（2×SSC，0.1％吐温20），并孵育15分钟。除去缓冲液2，并在缓冲液3 FO洗大脑（0.2×SSC，0.1％吐温20）-R 30分钟，重复此步骤2的两倍。由缓冲器4（0.1×SSC，0.05％吐温20中的PTW）更换缓冲液3中，温育5分钟。 </li><li>除去上清液，并在500μl的PTW的在室温洗5分钟。 </li><li>在2％琼脂糖（在1x PBS中）嵌入到大脑和切vibratome部分（50-100微米）所描述的免疫组织化学。 </li><li>洗涤1X 5分钟，在加入500μl封闭缓冲液（1×PBS，0.1％吐温-20，0.2％（重量/体积）牛血清白蛋白，1％（体积/体积）的DMSO）。 </li><li>除去上清，加入500微升封闭缓冲液中2小时，在室温下。 </li><li>除去上清液，加入300微升抗DIG AP偶联抗体阻断缓冲液稀释1:4,000。 </li><li>孵育O / N在4°C。 </li></ol></li><li> NBT / BCIP染色<ol><li>洗5×15分钟，用缓冲PTW在RT。 </li><li>在染色缓冲液冲洗1X。洗两次5分钟的染色缓冲液（100mM的Tris /盐酸pH为9.5，50毫摩尔MgCl 2，100 mM氯化钠，0.1％Tween-20中）。 </li><li>除去上清液和染色用500μ升染色溶液（3.5微升4 - 氮蓝四唑氯化物（NBT）和每1ml染色缓冲液5 - 溴-4 - 氯-3 - 吲哚基 - 磷酸（BCIP））。 </li><li>停止冲洗3至5倍与PTW染色。 </li></ol></li></ol></li><li>安装部的<ol><li>使用的水溶性，非荧光封固剂（见材料表）安装在载玻片上的章节。 </li><li>保持幻灯片在冰箱4℃注:染色是稳定数周甚至数月。 </li><li>根据分析的化合物或共聚焦显微镜幻灯片。 </li></ol></li></ol>

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<li>Kizil, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+neurogenesis+from+neural+progenitor+cells+requires+injury-induced+expression+of+Gata3%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Regenerative neurogenesis from neural progenitor cells requires injury-induced expression of Gata3.</a> Dev Cell. 23, 1230-1237 (2012).</li>
<li>Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+the+adult+zebrafish+brain+from+neurogenic+radial+glia-type+progenitors%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.</a> Development. 138, 4831-4841 (2011).</li>
<li>Kyritsis, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+inflammation+initiates+the+regenerative+response+in+the+adult+zebrafish+brain%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Acute inflammation initiates the regenerative response in the adult zebrafish brain.</a> Science. 338, 1353-1356 (2012).</li>
<li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+of+organs+from+the+adult+zebrafish%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Dissection of organs from the adult zebrafish.</a> J Vis Exp. (37), (2010).</li>
<li>Shin, J., Park, H. C., Topczewska, J. M., Mawdsley, D. J., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+cell+fate+analysis+in+zebrafish+using+olig2+BAC+transgenics%5BTitle%5D)+AND+%22Methods+Cell+Sci%22%5BJournal%5D)">Neural cell fate analysis in zebrafish using olig2 BAC transgenics.</a> Methods Cell Sci. 25, 7-14 (2003).</li>
<li>Armant, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide%2C+whole+mount+in+situ+analysis+of+transcriptional+regulators+in+zebrafish+embryos%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Genome-wide, whole mount in situ analysis of transcriptional regulators in zebrafish embryos.</a> Dev Biol. 380, 351-362 (2013).</li>
</ol>

我们在这里描述的方法通过在成年斑马鱼的头骨推针诱发端脑损伤，并分析采用免疫组化和原位杂交的细胞和分子反应，这种损伤。这种技术优于其他现有的方法的主要优点是它的简单性，速度和成本效率。因此，这种技术，它允许生产许多受伤的大脑在很短的时间，可以很容易地结合大规模原位杂交筛选，以鉴定基因在再生和神经发生的作用。 在其上执行的损伤成年斑马鱼的年龄是至关重要的。如果鱼是年长超过1.5年，头骨太硬并有可能是颅骨被按下或断裂而不是在清洁穿孔。相反，在年轻的鱼（未满4个月），仍然有很多的神经回事，这可能LEAd来就反应神经发生假阳性结果。 在这种方法的另一个关键步骤就是验证刀伤的效率和排除两个半球都受伤。这可以通过染色的切片用抗-PCNA抗体进行监测。在介绍正确病变，增殖细胞核抗原应显著上调只有一个半球。 该诱导的机械性损伤都会有一些副作用，如非特异性细胞消融，因为次级变性，炎症和破坏的血 - 脑屏障的增加的细胞死亡，以及心室区的损伤和重要的是要记住脑脊液的一个可能后果流入脑实质。然而，受伤的鱼似乎从身体损伤非常小。在2分钟内，他们可以自由的游泳一次正常摄食行为是在大约4小时恢复。 3周后的我njury是形态学上不再可见。在我们的手中，从几百手术我们没有尽管事实上，我们有一个100％的伤害率5天后病变评估的组织学失去了一条鱼。 在过去几年中，如转基因方法等技术已被开发在斑马鱼产生的组织或细胞类型特异性的消融2。尽管这些技术是非常优雅和微创它们都是基于复杂的DNA结构，并建立几个稳定的转基因斑马鱼线，可以是非常繁琐和费时的产生。因此，简单的机械方法，如在本协议中所述的测定保持学习成人的大脑和神经再生的重要工具。

哺乳动物具有非常有限的容量成年生活中，以产生新的神经元，并在大脑成年神经主要限制在位于侧脑室的脑室下区（SVZ）2端脑区域，并且齿状的颗粒下层区（SGZ）回海马1。哺乳动物也不能修复因神经退行性疾病，中风或损伤大脑的损伤，有效。丢失的神经元将不会被替换，而是扩散不同类型的神经胶质细胞，包括星形胶质细胞，小胶质细胞和少突胶质的，是观察。由于这种增殖过程称为反应性神经胶质增生的结果，受伤的脑组织是由神经胶质疤痕，从而抑制神经元和轴突再生2-4的形成密封。 相反，哺乳动物，硬骨鱼类，如斑马鱼在成年阶段丰富地形成新的神经元，并具有高的再生和proliferat潜在的ive修复中枢神经系统2,5-7的病变。在成年斑马鱼大脑中包含的整个生命过程中8-11产生大量新神经元的16个不同的神经干细胞小生境。出生在成年斑马鱼大脑的这些特异性增殖区的神经元移植到自己的目标地区，他们将在那里成熟，融入现有的神经网络8,10,12-15。 其中在成年斑马鱼中确定的祖区，在端脑脑室区研究最多的是神经干细胞小生境之一。在这个特殊的祖细胞可分为三种不同的类型就其形态，分裂速度和不同的神经胶质或神经元标记物的表达。 I型和II型细胞样细胞具有长流程的放射状胶质。他们表达干细胞标志物，包括GFAP，巢蛋白和S100β。 I型细胞不增殖，而II型细胞proliferatË慢慢地，你可以看到它们的增殖标志物，如增殖细胞核抗原（PCNA）和desoxythymidine类似物的掺入表达。 III型细胞快速分裂的细胞，致力于成为神经祖细胞和神经元表达的标记基因，如PSA-NCAM 5,16。 虽然硬骨鱼的再生能力是有据可查的，只有很少的出版物已经描述和详细研究涉及在成年端脑神经元再生响应于损伤15,17-22的细胞和分子机制。破译牵连的成年斑马鱼中枢神经系统的再生和修复机制，并了解组成和再生神经之间的相似性和差异，我们开发了成人端脑损伤的斑马鱼模型。在这里，我们将介绍如何在视觉上在端脑hemisph之一产生伤害ERES用注射器针的帮助下，同时保持对侧未病变的一侧作为内部对照。我们还将展示如何监测细胞增殖和免疫组化基因表达和原位杂交技术检测损伤后的变化。

<table><tbody><tr><td>Tricaine</td><td>Sigma</td><td>335.2</td><td></td></tr><tr><td>10x DPBS (-/-)</td><td>Life technologies</td><td>14200-083</td><td></td></tr><tr><td>30 G syringe (Omnican 40)</td><td>B.Braun Melsungen</td><td>916162</td><td></td></tr><tr><td>Polyethylene molding cup tray&amp;nbsp;</td><td>Polysciences, Inc</td><td>17177C-3</td><td></td></tr><tr><td>PeqGOLD Universal Agarose&amp;nbsp;</td><td>Peqlab</td><td>35-1020</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Biochrom</td><td>W 2835919108</td><td></td></tr><tr><td>Aqua Poly/Mount</td><td>Polysciences, Inc</td><td>18606-20</td><td></td></tr><tr><td>Tween 20</td><td>Carl Roth</td><td>9127.1</td><td></td></tr><tr><td>DMSO</td><td>Carl Roth</td><td>A994.2</td><td></td></tr><tr><td>Dissection needle</td><td>Carl Roth</td><td>KX93.1</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Carl Roth</td><td>335.2</td><td></td></tr><tr><td>Loctite 414</td><td>Sch&amp;ouml;ffler und W&amp;ouml;rner</td><td>&lt;a href=&quot;http://62.157.117.162/BP2NAV500/ItemDetail_NAV.aspx?ItemID=06%201362%200020&quot;&gt;06 1362 0020&lt;/a&gt;</td><td></td></tr><tr><td>Glass slides</td><td>Labonord</td><td>5305103</td><td></td></tr><tr><td>Cover slips 24 x 60 mm</td><td>Labonord</td><td>5305060</td><td></td></tr><tr><td>Petri dishes, 94 mm</td><td>Greiner bio-one</td><td>632180</td><td></td></tr><tr><td>Falcon tubes, 15 ml</td><td>Greiner bio-one</td><td>188261</td><td></td></tr><tr><td>24-well plate</td><td>Greiner bio-one</td><td>662160</td><td></td></tr><tr><td>Anti-S100 (1:500)</td><td>Dako</td><td>Z031101-2</td><td></td></tr><tr><td>Anti-PCNA (1:400)</td><td>Dako</td><td>M087901</td><td></td></tr><tr><td>Anti-GFP (1:1,000)</td><td>Aves Labs</td><td>GFP-1020</td><td></td></tr><tr><td>anti-Dig AP-coupled antibody&amp;nbsp;</td><td>Roche Diagnostics</td><td>11093274910</td><td></td></tr><tr><td>5-Bromo-4-chloro-3-indolyl-phosphate (BCIP)&amp;nbsp;</td><td>Roche Diagnostics</td><td>1383221</td><td></td></tr><tr><td>4-Nitroblue tetrazolium chloride (NBT)&amp;nbsp;</td><td>Roche Diagnostics</td><td>1383213</td><td></td></tr><tr><td>Proteinase K</td><td>Roche Diagnostics</td><td>1092766</td><td></td></tr><tr><td>Polyethylene molding cup tray&amp;nbsp;</td><td>Polysciences</td><td>17177C-3</td><td></td></tr><tr><td>Vibratome VT1000S</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal microscope Sp5 DM6000</td><td>Leica</td><td></td><td></td></tr><tr><td>Dissecting microscope Nikon SMZ 645</td><td>Nikon</td><td></td><td></td></tr><tr><td>24-well flat bottomed tissue culture plates&amp;nbsp;</td><td>Falcon catalog&amp;nbsp;</td><td>353226</td><td></td></tr><tr><td>Student Dumont #5 Forceps</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>&lt;em&gt;Danio rerio Tg(olig2:EGFP)&lt;/em&gt;</td><td>Shin &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;24&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>&lt;em&gt;Danio rerio&lt;/em&gt; WT strains (&lt;em&gt;e.g.&lt;/em&gt;, AB2O2)</td><td></td><td></td><td></td></tr></tbody></table>

stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration

成年斑马鱼具有惊人的能力，损伤后再生的中枢神经系统。为了研究细胞响应和参与斑马鱼成人中枢神经系统（CNS）的再生和修复的分子机制，我们开发了成年端脑损伤的斑马鱼模型。 在这种方法中，我们通过手动推胰岛素注射器针头插入成年斑马鱼端脑产生伤害。在不同的岗位有伤天和，鱼牺牲了，他们的大脑解剖出来，并通过免疫组化和/或原位杂交（ISH）用适当的标记，观察细胞增殖，gliogenesis，和神经染色。对侧未病变的半球作为内部对照。这种方法相结合，例如用RNA深度测序可以帮助筛选新基因在成年斑马鱼端脑神经，再生和修复的作用。

使用本文中所描述的工作流程，病变被引入到成年斑马鱼端脑（ 图1A-1B）的右半球。正确的伤人导致病变的运河，从背部延伸到腹部穿过端脑（ 图3C）的整个大脑皮层。病变应限于半球之一，并且不应越过内侧心室。病变运河与亮视野显微镜（ 图3C）可见后约35日15就会痊愈。 这是以前表明，伤人的上调增殖细胞核抗原（PCNA; 图3A）后，在3至7 DPL和S100β（ 图3B）是通过检测免疫组化15。 PCNA和S100β的染色是重叠的，这表明放射状胶质细胞的增殖。 F鱼24:urthermore，以可视化少突胶质前体细胞（OPCs的），刺伤测定法对Tg的（EGFP OLIG2）执行。后用抗GFP抗体染色，在靠近病变运河原花色素的积累是可检测的（ 图3D）。这种积累是短暂的，和OPC集群不是在35 DPL 15观察了。 如果病变没有出台不当，病变运河将不可见，并上调PCNA和S100β或原花色素的积累也不会察觉。 PCNA是最敏感的指标，脑损伤。为了验证该刀刺伤的效率和排除两个半球都受伤，强烈建议始终染色切片用抗PCNA抗体。在正确地导入病变，增殖细胞核抗原应该是显著上调（高达4倍的15）中唯一的半球之一。虽然在大多数情况下，它是足以只使用对侧未病变的一侧作为内部控制，以监测基因表达的变化，强烈建议到基因表达的控制半球与野生型大脑半球端脑的表达比较。以这种方式，可以检测基因表达的病变会影响两个半球的任何全身作用。 该刀伤检测与转录调节（TRS）的表达系统屏幕的组合（25和Schmidt 等人 ，未发表）也可以用来识别TR基因在神经发生和再生（ 图4A-4C）可能发挥的作用。 这种方法已经确定了一些所表示的端脑和其表达特异性上调响应于损伤（ 图4A-C）的因素。 <imgALT ="图1"FO:内容宽度="5英寸"SRC ="/ files/ftp_upload/51753/51753fig1highres.jpg"宽度="500"/&gt; 图1:成年斑马鱼端脑刺伤人示意图。 A）的胰岛素注射器针头是通过一个成年斑马鱼的头骨推到生成端脑损伤。白色的虚线表示两个端脑半球之间的边界和绿色十字表示成年斑马鱼大脑的刀伤B）背视图的位置。主要的脑部区域表示。绿十字显示性损伤。C）针，其作用是诱导端脑损伤，尖端测量2毫米的位置。嗅球（OB），端脑（电话），并视顶盖（OT）。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51753/51753fig2highres.jpg" width="500" /> 图2 S。成年斑马鱼大脑中修剪琼脂糖块电气原理表示之前vibratome切片。前到了。嗅球（OB），端脑（电话），视顶盖（OT），小脑（CB）和髓质（M）。比例尺:500微米。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51753/51753fig3highres.jpg" width="500" /> 图3。再生反应一刀，受伤。 AD）的成年斑马鱼脑5天后病变（DPL）。AB）的增殖标志物PCNA（A）和放射状胶质标志物S100β（B）是在刺伤人上调在脑室区（箭头）。 （ 三）损害通道是在明亮的视场照明（箭头）可见。脑室区由绿线表示。 （ 四）少突胶质前体细胞（OLIG2:EG指纹+）积聚在病变（箭头的网站）。比例尺:200微米。在所有面板，右半球是损毁半球。背到了。中的虚线A到D表示的大脑皮层和subpallium之间的边界。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51753/51753fig4highres.jpg" width="600" /> 图4。基因上调响应刺伤害的例子。注意eomesa（A，B）和基因Sox4a（C）在右侧端脑半球mRNA的表达（捅半球）的强烈上调（箭头）作为监测在 5 DPL 原位杂交。左半球是没有受伤，并作为控制。 （b）表示A。背背端脑区域的高倍放大到了。比例尺:200微米的A和C和55微米的为B的虚线A和C表示的大脑皮层和subpallium之间的距离。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51753/51753fig4highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <table><tbody><tr><td colspan="2">缩略语</td></tr><tr><td>美联社</td><td>碱性磷酸酶</td></tr><tr><td> BCIP </td><td> 5 - 溴-4 - 氯-3&#39;-indolyphosphate </td></tr><tr><td> BSA </td><td>牛血清白蛋白</td></tr><tr><td>中枢神经系统</td><td>中枢神经系统</td></tr><tr><td>挖</td><td>地高辛</td></tr><tr><td> DMSO </td><td>二甲基亚砜</td></tr><tr><td> DPL </td><td>天后病变</td></tr><tr><td> eomesa </td><td> 脱中胚蛋白同源物一 </td></tr><tr><td> GFAP </td><td>胶质纤维酸性蛋白</td></tr><tr><td>小时</td><td>小时</td></tr><tr><td> HYB缓冲</td><td>杂交缓冲液</td></tr><tr><td> ISH </td><td> 原位杂交</td></tr><tr><td>甲醇</td><td>甲醇</td></tr><tr><td> NBT </td><td>硝基四氮唑蓝</td></tr><tr><td>原花色素</td><td>少突胶质前体细胞</td></tr><tr><td> PBS </td><td>磷酸盐缓冲盐水</td></tr><tr><td>增殖细胞核抗原</td><td>增殖细胞核抗原</td></tr><tr><td> PFA </td><td>多聚甲醛</td></tr><tr><td> ProtK </td><td>蛋白酶K </td></tr><tr><td> PSA-NCAM </td><td>多唾液酸神经细胞黏附分子</td></tr><tr><td> PTW </td><td> PBS，0.1％Tween-20的</td></tr><tr><td> RNA </td><td>核糖核酸</td></tr><tr><td> RT </td><td>室温</td></tr><tr><td> SGZ </td><td>颗粒下层区</td></tr><tr><td> 基因Sox4a </td><td> 含4A基因SRY-盒</td></tr><tr><td> SSC </td><td>柠檬酸生理盐水钠</td></tr><tr><td> SVZ </td><td>脑室下区</td></tr><tr><td>红素</td><td>转录调节</td></tr></tbody></table>

Watch this Scientific Journal Video about Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration at JoVE.com

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

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15.3K 次观看.  Karlsruhe Institute of Technology. To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

视频: Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

斑马鱼高血糖的急性和慢性模型:评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

科研

发育生物学

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

神经科学

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 ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

<meta charset="utf8"></head><body>用于再生和发育研究的稳健斑马鱼细胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>作者聚焦:绘制发育和再生过程中斑马鱼神经系统中的细胞连接图

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

胚胎和幼体斑马鱼的高分辨率细胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生

摘要

探索更多视频

斑马鱼高血糖的急性和慢性模型:评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

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

成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法

在斑马鱼幼虫脊髓横断

基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

摘要

探索更多视频

斑马鱼高血糖的急性和慢性模型:评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

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

成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法

在斑马鱼幼虫脊髓横断

基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生