这项工作由美国国立卫生研究院资助DP2OD008470部分得到了支持。另外，RAM是部分由圣母研究生院的大学提供了资金支持。我们感谢生物科学系，中心斑马鱼研究，中心的员工干细胞与再生医学在Notre Dame大学。我们特别感谢实验室的成员参与有关肾脏生物学及其对这项工作有帮助的反馈讨论。

对于在本协议中所描述斑马鱼胚胎工作的过程被机构动物护理和使用委员会在Notre Dame大学的批准。 1.溶液的制备<ol><li>通过在RT混合73.0克氯化钠，3.15克氯化钾，9.15克CaCl 2，并在5升蒸馏水9.95克硫酸镁 ，并存储使E3胚胎介质的50倍原液。 </li><li>对于斑马鱼胚胎的培养中，稀释的E3胚胎媒体库存的50倍原液用蒸馏水1X工作液，再加入200微升的0.05％的亚甲基蓝的每1升1倍的E3作为杀菌剂，并储存在RT。 </li><li> 40毫升50×E3库存与0.06毫克PTU组合使E3胚胎媒体与0.003％1-苯基-2-硫脲（PTU）一个色素沉着封闭溶液。然后使体积到总共2L的蒸馏水。 </li><li>炒出的E3 / PTU O / N在室温得到PTU粉成溶液。然后存储E3 /在PTURT。 注意:E3 / PTU具有大约一周的保质期，和更老的解决方案将是在斑马鱼幼体阻断色素发展不太有效。 </li><li>通过加入1g三卡因到500毫升蒸馏水使0.2％三卡因（MS-222）的麻醉剂溶液并用1M Tris，pH值9.5将pH调节至7.2。 </li><li>制备通过将1×E3（无亚甲蓝）的溶液中，使用于成像的2％的甲基纤维素溶液中，用0.2％三卡因加入在4℃1/10体积。 注意:甲基纤维素溶液是用来轻轻稳定显微镜活胚胎厚，透明的溶液。操作被执行后的胚胎可以在1×E3洗涤以溶解甲基纤维素不损伤动物并在同一检体后续阶段使显微镜。 </li><li>时冷却至4℃，剧烈搅拌在搅拌盘1倍E3 /三卡因溶液逐步添加甲基适量同时继续纤维素粉末剧烈搅拌。逐渐将粉末添加到该溶液中，以防止不溶性甲基纤维素聚集体的形成。 </li><li>所有的粉末混合到2％甲基纤维素/ E3 /三卡因溶液后，搅拌在4℃冷室O / N复合材料解决方案。 注:低温是最好的粉末溶解完全。如果一个晚上后，解决的办法是不明确的，搅拌额外的工作日和/或第二O / N。 </li><li>从2％甲基纤维素/ E3 /三卡因溶液，等分试样的混合物在4℃下30分钟或高达2小时除去气泡进入以高速（12 000 XG）2.4毫升管和旋转。 </li><li>放置在4℃的2％甲基纤维素/ E3 /三卡因溶液长期储存。 注意:在此之前，用法，等份应预先温热至RT用于与斑马鱼标本工作。 </li></ol> 2.工具的制备<ol><li>准备一个胚胎操纵的工具，用于采购订单sitioning胚胎显微注射，通过连接在一个5"直解剖针的端部的凝胶加载提示，并用胶带或强力胶固定。 </li><li>首先将火准备用针管注射拔针头，抛光10厘米硼硅玻璃长丝放入仪器，并拧紧处理安全硼硅玻璃。 </li><li>拉硼硅玻璃时尚细锥形针。使用以下设置:加热540，拉245，速度200和125的时间。 </li><li>培养皿内放置针，上橡皮泥条暂停，以保护他们的针尖，并保持覆盖，防止灰尘堆积的菜。 </li><li>完成制备用于显微注射的针，用剃刀刀片或细镊子边缘切针的拔出端以创建一个尖锐的角注射针尖的直径为约0.05-0.1毫米。 </li><li>为了使显微注射托盘，准备在一个10 1.5％琼脂糖/ E3溶液0毫升锥形瓶中，并通过在微波的E3加热到沸腾，然后允许在室温冷却约10分钟，溶解琼脂糖。 </li><li>倒入冷却的琼脂糖/ E3溶液放入培养皿，使基础具有大约0.5cm的深度。当这已固化的倾具有大约0.3厘米的深度的第二层，并插入预制胚胎以及模具和允许琼脂糖在RT固化。 注意:当插入模具插入顶层琼脂/ E3层，将所述模具放入琼脂以一个角度以减少气泡的数目。 </li><li>当整个注射托盘已成立，抬预制胚胎以及塑造出缓慢而小心地使用scoopula，然后填写与E3的解决方案和存储的菜在4℃。 </li></ol> 3.胚胎制备方法<ol><li>通过将分隔成交配室设立斑马鱼交配坦克，并与系统中的水满。 </li><li>放置一个鱼分频器的S每侧UCH每个交配室包含一雌一雄鱼，并覆盖每个配合腔。 </li><li>第二天早晨，取出分隔，使产卵。 </li><li>检查鱼后约30分钟，并返回成年人水族馆后，通过传递配合水箱水通过细筛网过滤工具收集他们的胚胎。 </li><li>倒置滤网过培养皿和E3冲洗收集胚胎。 </li><li>在28.5°CO / N孵育胚胎。 </li><li>当胚胎已达到24-26体节阶段26，倒出大部分E3介质中，然后dechorionate加入100微升的50毫克/毫升的链霉蛋白酶胚胎的每个培养皿大约15温育在室温（23℃）的菜分钟。 注意:这将需要约24-25小时，从受精的时间，如果他们被收集在方式描述并在28.5℃培养O / N胚胎达到24-26体节阶段。 </li><li>装满菜E3然后轻轻漩涡撞出从胚胎绒毛膜。 </li><li>倒出E3 /链霉，用25毫升新鲜E3漂洗菜。重复冲洗步骤去除所有链霉。 </li><li>要阻止色素沉着的发展，倒出E3并用25毫升新鲜的E3 / PTU的时候胚胎已经达到24小时受精后更换。 注意:对于实验跨越数日，每日更换一次新鲜的媒体E3 / PTU解决方案，以保持干净的菜。 </li></ol> 4.肾毒素显微注射的解决方案<ol><li>关于注射的当天，制备用适当的车辆控制沿着所需肾毒素溶液。 </li><li>涡流或轻轻混匀，以确保药物（s）是/都是在溶液中，检查水柱的管和顶部的两侧。 注意:肾毒素溶液可以制备成含有荧光缀合的葡聚糖的痕量监测显微注射效率和也在subseq评估肾清除uent时间点20,28。 </li><li>通过螺纹细凝胶加载提示到针的后部装入〜2-3微升肾毒素的修整显微注射针和用一块胶带的垂直暂停针以使该溶液通过重力来填充修整针尖。 </li><li>一旦针尖是满的，固定在显微加载针。 </li><li>通过将矿物油的液滴到微米滑动测试注射体积和注射进油来评价液滴尺寸。 500复注射体积的直径为0.1毫米。 </li><li>除去从培养箱胚的培养皿，并通过加入约5mL的0.2％三卡因的对胚胎菜麻醉。 </li><li>为了确保完全麻醉，轻轻地用胚胎操纵工具的尖端碰触胚胎。缺乏运动的指示充分麻醉。 </li><li>麻醉成功后，转移胚胎到注塑模具用TRANSF呃吸管。 </li><li>机动每个胚胎成不同以及模具放置头井使得行李箱沿凹陷休息和尾巴竖起走出抑郁的最深处的区域。 </li><li>将填充进针显微和针尖位置，旁边是一个胚胎。 </li><li>轻轻地向前移动操纵杆针头插入尾容器并踩下脚踏板提供显微注射。 注:成功注入可以通过观察液体来衡量进入流通。另外，用荧光结合的葡聚糖的nephrotoxicant的共注射使研究者来验证到程序随后成功注入。 </li><li>轻轻地向后拉操纵杆，从胚胎中取出针。 </li><li>注射后，将胚胎转移到一个干净的菜和冲洗以除去三卡因，并用新鲜的E3 / PTU替换。 </li><li>孵育胚胎到所需的时间点到驴小号的形态，其可通过摄影在甲基纤维素安装介质，或处理根据需要被记录为实验分析。 注:胚胎的复苏应该评估，以确定个人的水肿，其中常见的指示肾损伤的百分比。 </li></ol>

The authors have nothing to disclose.

治疗药物的不同数量已与AKI 29有关。已经出现了在理解由许多单独的化合物，如氨基糖苷类庆大霉素30和广泛使用的化学治疗顺铂31,32引起的损伤显著研究进展。参与这些条件下一些病理变化，但是，仍正在进行的研究的主题。一个新兴的挑战仍然是如何理解多种药物产生不利影响的患者，高危人群尤其是那些如在重症监护设置和老人29。因此，模式，使关于药物诱导的AKI的效果进一步蜂窝基础的研究服务改善这种状况的检测和欣赏动态药物相互作用中起重要作用。 哺乳动物模型，如小鼠和大鼠的工作已经积分建立在肾脏以下AKI细胞变化，卜t的一限制这些系统是直接，实时的观察是由于建筑复杂性和脏器内部的位置的限制。简单地说，肾脏不适用于直接观察，这需要个人被牺牲来分析肾。这些限制可通过使用斑马鱼的胚胎，其形成有与其他脊椎动物，包括人33的保守区段结构中的两个肾的简单肾脏被抵消。斑马鱼发育发生子宫外 ，并有轻微的色素沉着，允许外部线索34的肾器官和响应监测的直接观察。用于分子分析工具，如基因表达的35-37，已经利用具有高分辨率，以确定nephrogenesis 38-40的特性。通过化学遗传学的小分子检测实验操作已经非常成熟的斑马鱼和适用于kidnEY 41-43。事实上，在最近几年，斑马鱼已经被应用到模型的代理从对乙酰氨基酚到真菌毒素和马兜铃酸44-48肾毒性。要注意，也有在斑马鱼胚胎的AKI建模显著的局限性，因为肾结构的在这个阶段中发展的非常简单的是重要的。例如，斑马鱼不适合于寻址肾组织被密集地填充有由一个复杂的基质包含多个间质细胞类型包围肾内多因素的相互作用，在啮齿动物或人的后肾的情况。因此，斑马鱼是最适合AKI期间可视化的自主肾细胞和分子的变化。 这里所描述的显微注射的方法，同时具有陡峭的学习曲线，是研究都发育和疾病状态，包括肾毒性非常有用的。这种技术可以用来对TE圣药物单独使用或组合使用。此外，注射剂可以在宽范围的开发时间点中进行。在斑马鱼幼体进行静脉内显微注射的类似，同样可行的方法已经由COSENTINO 等人 21先前已经描述。在他们的方法之一显著区别，相比，这里描述的方法是，胚胎被注入固定利用保持吸管21。使用保持吸管的是一个可行的替代的注塑模具。研究人员谁寻求实现显微注射作为nephrotoxicant代理交付方式应该意识到这个替代不妨来比较的方法来确定哪些是个人可取，因为学习操纵控股吸管，以免破坏胚胎将涉及实践只是因为它需要实践来成功地操纵和使用简单的注塑模具有凹陷腔体的电磁制动microinjectYOS。 当执行此过程，重要的是制备适当大小的注射针头，和削减对针尖的角度，以优化光滑的入口和出口到胚胎循环。在注射过程中，关键是要监视注射体积以评估样品之间的肾毒素递送的一致性。如果可以的研究者是不确定的量的变化而进行实验来进行用千分尺注射量的定期评估。例如，由于技术的性质，针尖可部分或完全用细胞碎片堵塞。这种并发症可以通过周期性地清除针，以确保所喷射流体的一致性来抵消。此外，像酚红的重要染料可以加入到该混合物中以充当注入的视觉标记和协助监测流体分散49,50。此外，示踪分子的注射可以有助于可视特定细胞群，在注射以下。荧光葡聚糖部分，具体为10 kDa的葡聚糖缀合物的共注射，有许多应用16,17。在这种情况下，荧光强度的评估可以紧跟确认具有最小泄漏成功显微注射的方法进行。强度可使用适当的图像捕捉摄影，然后在随后的时间点复查以测量荧光强度的变化，以便测量肾清除51来测量。另外，在近端小管的葡聚糖的重吸收提供此肾段16,17的功能的代理。 两者合计，有显微注射可以用于调查AKI与斑马鱼，特别是该模型提供了机会，以解决在体内药动学许多方面。因此，一旦掌握，肾​​毒素显微注射入斑马鱼胚胎为研究肾有益的范例。

AKI是肾功能可能导致灾难性的健康后果1的突然损失。 AKI是一个显著的医疗保健问题，全世界由于约20％的住院患者中的发病率很高，在重症监护的情况下30-50％甚至更高的速率和老人，以及50-70％的1-3死亡率。不幸的是，AKI的发病率不断上升，预计未来十年进一步升级，部分原因是由于多种因素的多样性，可诱发急性肾损伤，包括手术后应激，缺血和接触到的肾毒性，如抗生素和化疗药物4。 AKI涉及肾脏内突细胞损伤，在肾，这是必要的功能单元，并且是由一个血液过滤器和排出尿进入中央集合管1的分段小管通常发生。当肾的一个显著数目是AKI过程中被损坏，直接影响包括从循环通过从死亡和垂死的细胞1由于阻塞肾单位在废弃物清除中断，并减少或废除流体流动。随着时间的推移，肾小管阻塞可导致肾整，永久地降低了肾功能1退化。在肾脏生理改变以下的AKI也涉及复杂的炎性事件，可导致慢性结疤1。 尽管有这些成果，肾有一定能力的AKI的进行重构肾小管上皮细胞5,6后接受再生。虽然出现了肾再生的分子越来越了解，该机制仍然在许多方面难以捉摸和必要继续调查7。在何种程度AKI导致永久性肾损害也仍然不明。目前的研究表明，对肾脏的再生潜能是最高以下AKI的较不严重的情况下，而更明显或反复发作导致慢性肾脏病（CKD）和终末期肾病（ESRD），需要救生移植或透析8,9-高潮。此外，个人从CKD已经患有处于收缩AKI 8,9的严重发作的风险更大。两者合计，很明显，持续的基础和临床研究是至关重要的理解，治疗和预防AKI。 研究与动物模型已经在欣赏AKI 10期间发生的本地和环境的改变的进展工具。为了扩大这种认识以及开发新的疗法，斑马鱼的动物模型已经在以各种方式11,12使用。斑马鱼肾脏的肾单位，在胚胎和成人两种，显示与哺乳动物13-16高度保护。另外，在泽肾上皮损伤brafish类似于高等脊椎动物，由此肾小管细胞的局部破坏之后是管内增殖和肾结构17-19的重建过程。在胚胎，但是，从像顺铂的肾毒性广泛肾小管损伤与杀伤力20,21相关联。相比之下，成年斑马鱼生存AKI并表现在肾脏实质性的再生能力。例如，在暴露于氨基糖苷类抗生素庆大霉素，斑马鱼再生肾小管上皮损伤，增加新的肾单位以及22-24。虽然这些庆大霉素引起的AKI的研究提供了宝贵的资料，了解来自不同的肾毒性肾功能损害仍然至关重要升值的影响，并应对不同类型的损害25。 斑马鱼胚胎，由于其大小，透明度和遗传易处理性，对肾毒素的研究很多好处&lt;SUP&gt; 25，其中，用于管理调查的分子（多个）注射20,21的方法。肾是由24小时受精后（HPF）形成，并开始约48 HPF 26,27来过滤血液。因此，胚胎肾的迅速形成和功能便于实验分析。然而，显微注射的过程中具有技术挑战，并有可能是一个陡峭的学习曲线，以掌握该技术。在这个视频文章中，我们将介绍如何执行显微注射​​，并以提高成功的注射速度提供故障排除技巧。

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Chloride</td>
			<td style="width:229px;">American Bioanalytical</td>
			<td style="width:129px;">AB01915</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB01652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB00366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td>Aldrich Chemistry</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate (Tricaine)</td>
			<td>Fluka Analytical</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass</td>
			<td>Sutter Instruments Co.</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>Mo. P097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Diposable Petri Dishes, Sterile, Corning:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm x 15mm</td>
			<td>VWR</td>
			<td>25373-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm x 15mm</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;(microinjection tray) 150mm x 15mm</td>
			<td>VWR</td>
			<td>25373-187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Temperature Incubator</td>
			<td>Fischer Scientific</td>
			<td>11 690 516DQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Tweezer</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-5010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Micrometer</td>
			<td>Ted Pella, Inc.</td>
			<td>2280-24</td>
			<td></td>
		</tr>
	</tbody>
</table>

nephrotoxin microinjection in zebrafish to model acute kidney injury

肾脏是容易暴露在他们从血液中过滤化学物质伤害。这会导致与在肾功能和临床症状称为急性肾损伤（AKI）的发展迅速下降有关器官损伤。药剂用于治疗的医疗情况，从细菌感染到癌症，当单独给药或与其它药物组合，可以发起AKI。斑马鱼是一个有用的动物模型来研究对肾功能的化学作用在体内 ，因为它们形成由那些保守与高等脊椎动物，包括人类在内的肾功能单位的胚胎肾。此外，斑马鱼可以用于执行遗传和化学屏幕，这提供机会以阐明AKI的细胞和分子方面和开发治疗策略如肾保护分子的鉴定。在这里，我们将演示如何显微注射入斑马鱼胚胎可以用作用于肾毒素研究一个范例。

显微注射站设立包括立体显微镜，显微操纵器和压力调节器（ 图1A）。注入板的透优选此过程（ 图1B）中，以查看标本。注射针的制备涉及拉适当硼硅酸盐玻璃，然后通过制备用切割和最后背加载针的边缘。最佳地，针尖斜面，而不是在切割过程期间钝（ 图1C），这是由急剧斜着切割器械，边缘制成，例如细钳子的切口， （ 图1D）。这个斜面边缘是关键的，将极大地影响到针轻轻插入斑马鱼的能力。 上注射当天，胚胎使用坚固但柔韧胚胎定位操作工具（ 图1E）。对于注射，胚胎操纵，使得该躯干沿注入托盘的侧休息以提供用于随后的注射步骤（ 图1F）的杠杆作用。同时注入，应该着眼于胚胎躯干的角度平面的可视化的针插入血管目标，然后观察注射材料进入流通（ 图1G）。 斑马鱼胚胎的透明性有助于显微注射过程，并且可以进一步通过PTU的化学处理在24高倍视野增强一个典型的实验时间过程（ 图2A）中，以减少色素沉着的发展。这里，胚胎标本被允许通过72 HPF阶段发展，然后要么模拟注射，显微注射用生理盐水车辆，或具有2.5毫克/毫升庆大霉素（ 图2A）显微注射。随后现场观察，于一天和两天后喷射进行，使用透射照明用立体显微镜（ 图2B）。相比模拟或盐水注射的胚胎中，只有庆大霉素注射个体表现出水肿的发展（ 图2B），在这种情况下心包水肿，提示的正常肾功能与先前公布的观测11,20,21相一致的废除。 <img alt="图1" src="/files/ftp_upload/54241/54241fig1.jpg" /> 图1 的显微注射装置和工具。（A）的注入站设置与立体显微镜（左），显微和针保持器（中）和压力调节器（右）。注入板的（B）中透照。 （C）左图:冲裁针。右:斜面切割针。 （D）的FINE钳用于切割针尖。 （E）用于插入和试样放置到注射模具操纵的工具。 （F）胚胎摆着显微注射模具。比例尺= 0.5mm左右。 （G）的针的定位毗邻胚胎用于注射。比例尺= 0.15毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54241/54241fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54241/54241fig2.jpg" /> 图2.实验时间表和注射后水肿检测。（A）例肾毒素在斑马鱼实验时间表，在这里应用到庆大霉素研究。胚胎dechorionated并在24小时后受精（HPF）PTU溶液处理。每一天中，PTU溶液倾析，并用新鲜介质替换为小了指示r绿色箭头。在72 HPF，胚胎被麻醉，转移至注射模具中，然后进行如下处理:模拟注射或注射盐水（载体对照）或庆大霉素。在新鲜培养基振兴胚胎后，在1和2天后注射（96和120高倍视野，分别）用于分析和图像采集，观察胚胎。 （B）上图:未注射斑马鱼胚胎。中东:胚胎在72 HPF注射生理盐水。底部:胚胎注射2.5毫克/毫升庆大霉素。比例尺= 0.5毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54241/54241fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury at JoVE.com

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

从肾毒素，其中包括药品，从抗生素对化疗药物产生的肾损伤，可导致复杂的疾病，其发病机制尚未完全清楚。这个协议演示斑马鱼如何可以用于这些条件下，它可以被应用到的肾脏保护措施的识别疾病建模。

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

nephrotoxin-microinjection-in-zebrafish-to-model-acute-kidney-injury

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K 次观看.  University of Notre Dame. 从肾毒素，其中包括药品，从抗生素对化疗药物产生的肾损伤，可导致复杂的疾病，其发病机制尚未完全清楚。这个协议演示斑马鱼如何可以用于这些条件下，它可以被应用到的肾脏保护措施的识别疾病建模。 

视频: Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...