我们要感谢Iain Drummond博士和Vladimir Korzh博士分享肾GFP转基因株系。我们也要感谢纽特派团提供必要的资源进行这项工作。这项研究部分得到赠款的支持:K08DK082782，R03DK097443（NIH）和HSCI Pilot Grant（AV）。

本研究按照"国家卫生研究院实验动物护理与使用指南"中的建议进行。该协议由NYIT骨科医学机构动物护理和使用委员会（NYITCOM IACUC）批准。所有的手术和体内实验都是在三联麻醉下进行的，所有努力都是为了尽量减轻痛苦。 获取和维护胚胎<ol><li>通过穿过肾GFP荧光斑马鱼获得胚胎。 注:荧光标记的斑马鱼转基因株系的实例列于表1 。 </li><li>在受精24小时后，在E3溶液中将胚胎保持在28.5℃。 </li><li> 24小时后，用含有0.003％1-苯基-2-硫脲（PTU）的E3溶液代替培养基。 注意:使用PTU是因为它阻断了黑色素生成中依赖于酪氨酸酶的步骤从而防止色素沉着。这里，该解决方案被称为E3-PTU。 </li><li>在28.5°C下培养受精胚胎直至&gt; 6 dpf，此时肾脏已经成熟。 注意:最好在7-9 dpf窗口中使用幼虫。 </li><li>使用40X放大的荧光解剖显微镜和绿色荧光发射滤光片，选择具有明亮的GFP荧光的幼虫。 </li></ol> 2.安装斑马鱼进行实时成像<ol><li> 如果在3 dpf之前照射胚胎，请在安装斑马鱼进行成像之前从任何未孵化的斑马鱼中取出绒毛膜。 <ol><li>用镊子（优选）手动脱敏或用蛋白酶混合物进行化学处理。 注意:脱胶可以获得最佳的成像效果。 </li></ol></li><li>使用E3-PTU溶液冲洗培养皿中的绒毛膜碎屑，并用E3-PTU补充盘。 </li><li> 准备工作重复1-2％低熔点（LMP）琼脂糖与0.2mg / mL三卡因溶液，以安装斑马鱼进行肾摄影和活体成像。 <ol><li>如果LMP琼脂糖已经预先准备好了，再在微波炉中加热，使其熔化。 </li></ol></li><li>一旦制备了LMP琼脂糖，将35 mm塑料培养皿放在解剖显微镜上。在附近放置一个拉玻璃探头，以正确定向麻醉的幼虫。 注意:重要的是要及时做这个步骤，并将所有的东西都放在适当位置，因为胚胎必须在琼脂糖凝固约1-3分钟之前定向。 </li><li>在将胚胎转移到琼脂糖之前，确保琼脂糖是凉爽的（ 即在体温附近）。使用塑料或玻璃移液管将胚胎放入冷却的熔化琼脂糖三卡因溶液中。 注意:这是因为过热的琼脂糖可能不利于胚胎，导致心脏骤停或死亡</li><li>使用转移移液管，将琼脂糖溶液中的胚胎重新取出，并将胚胎/幼虫置于35毫米的中间。 </li><li>快速铺展含有胚胎/幼虫的琼脂糖均匀地覆盖35毫米的盘底;琼脂糖的最佳使用量为〜1-1.1 mL。 </li><li> 使用玻璃探针将胚胎定位在最佳位置进行光消融和成像。 注意:电影1显示单边光消融的胚胎/幼虫的最佳方向。这个步骤是最时间敏感的，应该在琼脂糖开始凝胶化之前快速进行，因为在凝胶开始后重新调整鱼的任何尝试都是有害的。 <ol><li>定向幼虫，使感兴趣的肾段垂直于束通道，而对侧段远离激光束（见影片1 ）。 注意:可以通过转动幼虫来实现，如电影1所示 。 </l我&gt; </ol></li><li>让琼脂糖凝固约15分钟。覆盖培养皿以尽量减少蒸发。一旦琼脂糖凝固，胚胎或幼虫就可以进行光消毒。 </li></ol>激光烧蚀<ol><li>操作共焦成像系统。 注意:使用参考的成像平台（参见材料表）时，推荐的设置包括40倍放大倍率，0.8 NA水浸透镜和第一个分色镜，可以对样品进行488和405 nm的照明。检测应在绿色通道上进行。 </li><li>将包含固定化斑马鱼的盘定位在共焦阶段。 注意:此过程针对使用40-60X水浸透镜的直立配置进行了优化。然而，应该可以修改倒置显微镜的程序。 </li><li>在直立的配置中，将包含胚胎的培养皿放在微观的顶部ope滑动，并使用造型粘土（ 例如，橡皮泥）将其保持在适当位置。将陪替氏培养皿的玻片放在共聚焦显微镜的台上。 </li><li>加入3毫升成像溶液E3-PTU（见步骤1.3）与0.2毫克/毫升三卡因。 注意:在将幻灯片放在舞台上之前，也可以立即添加成像解决方案。成像溶液的添加应该非常小心，以防止琼脂糖漂浮在盘底部。 </li><li>切换到水浸透镜（40或60X）。缓慢升起舞台，确保镜头稍微倾斜，以防止空气被束缚在光束路径中。一旦透镜以一定角度接触溶液，将其居中，使其处于胚胎的视野。 </li><li>使用荧光光源查找感兴趣区段。表面定位受伤的分支，以尽量减少光散射（见影片1 ）。 注:子序列t步骤使用引用的软件（参见材料表），但该过程可以轻松适应其他平台。 </li><li>打开软件导航到"查看"| "收购控制"| "C2plus Compact GUI"，将"像素停留时间"设置为"1.9μs"，"框架尺寸"设置为"512 x 512像素"，将"针孔尺寸"设置为"90μm"。将"405nm（或某些系统中的408nm）激光强度"设置为零，将"488激光强度"设置为"低"（优选&lt;1％）。调整"增益"以获得良好的动态范围，但不会使信号饱和。 注意:这些值并不重要，用于一致性。当使用405nm激光时，会导致GFP荧光增加。为了避免光漂白期间信号的饱和（步骤3.11），信号增益值应在绿色通道上按比例缩小。蓝色通道增益可以保持为零，因为它不用于sampling。 </li><li> 点击软件界面上的"扫描"，使用488 nm激光扫描肾脏。找到垂直于光束路径的感兴趣区段，并且具有良好的可视化，具有良好的GFP荧光。一旦确定了该区域，使用椭圆的感兴趣区域（ROI）绘制感兴趣的区域。 <ol><li>转到"ROI"| "绘制椭圆投资回报率"。 注意:这将用于在进行光漂白时持续测量平均GFP强度，从而允许给予精确剂量的激光治疗。 </li></ol></li><li>导航到"查看"| "收购控制"| "C2plus扫描区域"，并选择"带扫描区域"。矩形蒙版将出现在该界面内。使用光标手动定义矩形扫描窗口（ 即拖动，调整大小并转动掩码）以覆盖激光切割的目标区域。右键单击面罩区域接受选择。 注意:仅扫描所选区域。 </li><li>开始时间测量，同时使用488 nm激光监测绿色通道来激活GFP。确保488激光的强度相对较低（〜1％）。通过选择"Measure"|来测量感兴趣区域中GFP的平均强度"时间测量"。使用"图表"或"数据"选项卡来监视投资回报率内的平均强度值。 注意:采集速率由步骤3.9中设置的矩形窗口的像素驻留时间和大小定义，但通常可以快速监视信号（〜2-10帧/秒）。 </li><li> 通过将"激光功率"控制一直向右滑动，通过将强度增加到100％，用紫色激光（405nm）对肾细胞造成伤害。如果强度低，488nm激光器可以保持有效。 <ol><li>观察一行graph使用"图形"选项卡或GFP强度的数值使用"数据"选项卡，并等待它下降到所需的水平，例如基线的50％（ 如图1所示 ）。 注意:20 mW激光器已被用作参考激光单元的一部分（见材料表 ）;最大强度可能在系统之间变化。较低的强度可以通过更长时间的曝光来补偿（ 即使用百分比的GFP漂白作为总曝光量的度量）。 </li></ol></li><li> 一旦椭圆ROI（步骤3.8）中的GFP强度下降到所需的水平，通过将"激光功率"滑块一直向左移动，立即将紫色（405nm）激光的强度降低到"0"软件控制面板。 <ol><li>获得GFP荧光的新基线。通过获得X / Y的比例来确认消融。 注意: 见图1B; Y =消融前的平均强度，X =消融后的平均强度，使用ROI内平均4个连续样本。确切的目标比例（50％，60％ 等 ）取决于给定实验所需的损伤量。 </li></ol></li></ol>延时显微镜用碘化丙啶染色<ol><li>应用一般的时间流逝考虑（在之前的研究15中概述），以观察碘化丙啶染色，作为光消除后肾细胞损伤的相关性。 </li><li>如步骤2和3.7所述，将嵌入和光消除之前预孵育30μM碘化丙啶溶液（E3-PTU中的1％DMSO）3小时。 注意:在琼脂糖或成像溶液中不需要额外的碘化丙啶。 </li><li>诱导分期性肾损伤，如步骤3.1-3.12所述。 </li><li>获取时间流逝图像堆栈，作为描述在先前的研究中，使用488nm激光（GFP）和461nm激光（Propidium Iodide，PI）进行研究。 注意:两种颜色叠加在共焦点堆叠中，以关联GFP的消失和碘化丙啶染色的外观。 </li><li>设置获取延时图像叠层的参数如下:虚拟切片厚度= 4μm，z叠层间隔= 2μm，像素停留时间= 1.9μs，图像尺寸= 512 x 512，平均值= 2。 注意:这些参数允许连续进行10到15分钟的间隔记录最多4个幼虫，并确保样品的光漂白最小。 </li><li>使用与录制文件格式兼容的成像软件可视化和分析数据。 </li></ol>

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作者宣称他们没有竞争的经济利益。

应当注意，总体激光功率在系统之间变化。然而，使用百分比的GFP光漂白允许读出传递到荧光肾的总能量，而与激光功率的变化无关，并且由曝光长度补偿。但是，请记住，不同组织对这种光消融方法的反应有所不同。即使在肾成熟期间，在成年幼虫中获得GFP荧光在年轻胚胎中降低50％明显更难。在将该协议修改和适应不同应用时，应考虑到光消解的组织反应性的这些差异。因此，监测GFP荧光的?...

急性肾损伤（AKI） 1,2 ，也可称为急性肾功能衰竭，广泛定义为肾功能的突发性损害3 。虽然多年来对这种状况的了解程度有了显着提高，但发病率和死亡率仍然高达1,2 。目前对这种病症的治疗主要是支持性的，因为药物治疗的多个临床试验的结果为阴性4,5 。肾脏是独一无二的，因为它具有修复自身的能力。因此，AKI早期诊断后的支持治疗是限制发病率的最佳途径6 。然而，很难早期检测AKI，对于需要透析的患者，死亡率为惊人的50-80％ 5。随着肾脏修复自身的能力和缺乏这种病症的治疗方案，重要的是开发增强这种肾单位再生过程的方法。 AKI研究中已经有许多不同的模型，包括不同的伤害和动物模型。就肾脏损伤剂而言，氨基糖苷类抗生素庆大霉素已被用作导致AKI 7,8的肾毒性药物。然而，有几组发现庆大霉素治疗对斑马鱼胚胎是致命的9 。它造成管状损伤对于胚胎恢复来说太严重，使得再生的研究难以进行某种干预。哺乳动物模型，如小鼠和老鼠，也被认为是有价值的，但在AKI研究期间它们面临许多限制。啮齿动物模型的主要缺点也许是visua的困难使啮齿动物肾脏化，从而确定导致上皮细胞死亡和修复的精确时空过程。 约翰逊已经报道了基于激光消融的技术在胚胎和幼虫斑马鱼中诱导急性肾损伤9 。在用葡聚糖缀合物肌内注射后，他们使用脉冲激光消融来损伤肾脏。来自葡聚糖共轭物的荧光允许在小管上皮9中的损伤和再生的可视化。该模式克服了上述两个限制，但不允许分级伤害，难以在大型，任意细胞群上进行。 这里描述的新的基于激光消融的AKI斑马鱼模型解决了上述所有限制。幼虫斑马鱼中的肾肾是一种成熟的功能性器官，其中包含与哺乳动物ne相似的节段包括肾小球，近端和远端小管以及收集管10 。斑马鱼幼虫也是光学透明的，使得通过荧光技术观察肾脏是可行的。因此，斑马鱼是AKI的有价值的体内模型，幼虫肾肾（5-12天 - 受精后（dpf））可用于研究肾损伤和修复中涉及的细胞和分子过程。 本文提出了一种通过使用低能量（与脉冲激光系统）紫激光（405nm）相比较来表达特定的表达绿色荧光蛋白（GFP）表达的肾单位片段的方法。 GFP荧光允许靶向一组细胞，通过观察GFP光漂白使得发生可见的变化。此外，GFP（通过吸收紫光）作为能量吸收器来增强GFP表达肾细胞中的损伤。延时微型然后复制可以用来研究修复过程。研究发现细胞增殖，细胞迁移和细胞化生11,12,13都是可能在肾脏修复中发挥重要作用的潜在过程。然而，由于AKI现有模型的局限性，这些过程的相对重要性及其相互作用的细节难以揭示。使用这种新颖的方法，可以显示细胞迁移在急性损伤后的肾脏修复中起核心作用14 。

<table><tbody><tr><td>Petri Dishes, 35 x 10 mm</td><td>Genesee Scientific</td><td>32-103</td><td>Procedural Usage: Step 2.4, 2.7</td></tr><tr><td>Petri Dishes, 100 x 15 mm</td><td>Midwest Scientific</td><td>910</td><td>Procedural Usage: Step 1</td></tr><tr><td>Dechorination forceps - Electron Microscopy Sciences Dumont Tweezers 5 Dumostar</td><td>Fischer Scientific</td><td>50-241-57</td><td>Procedural Usage: Step 2.1.1</td></tr><tr><td>Plastic Transfer Pipet</td><td>Globe Scientific</td><td>135030</td><td>Procedural Usage: Step 2.5, 3.6</td></tr><tr><td>Tricaine</td><td>Sigma Aldrich</td><td>A5040-25G</td><td>Procedural Usage: Step 2.3, 3.4</td></tr><tr><td>Agarose</td><td>Fischer Scientific</td><td>BP165-25</td><td>Procedural Usage: Step 2.3</td></tr><tr><td>Pulled glass probe (manufactured manually from glass capillary tubes)</td><td>Fischer Scientific</td><td>21-1640-2C</td><td>Procedural Usage: Step 2.4</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1270</td><td>Procedural Usage: Step 1.5</td></tr><tr><td>SOLA Light Engine</td><td>Lumencor</td><td>SOLA SM-5-LCR-SB</td><td>Procedural Usage: Step 1.5</td></tr><tr><td>Eclipse C2 Plus Confocal Microscope System</td><td>Nikon</td><td></td><td>Procedural Usage: Step 3</td></tr><tr><td>1x E3 Solution</td><td></td><td></td><td>Recipe used to generate: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt; , 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt; Procedural Step Usage: 1.2, 1.3, 2.2, 2.3</td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td>Procedural Step Usage: 1.3, 2.2, 3.4, 4.2</td></tr><tr><td>NIS Elements Software</td><td>Nikon</td><td>C2+</td><td>Procedural Usage: Step 3</td></tr><tr><td>Laser Unit</td><td>Agilent</td><td>MLC 400</td><td>Procedural Step 3.11</td></tr><tr><td>Propidium Iodide (PI)</td><td>Sigma Aldrich</td><td>P4170-100MG</td><td>Procedural Step Usage: 4.2</td></tr></tbody></table>

precise cellular ablation approach for modeling acute kidney injury in developing zebrafish

急性肾损伤（AKI）是一种常见的死亡率高的疾病。具有肾脏的修复能力，可以在支持治疗后恢复足够的肾功能。然而，需要更好地了解如何在细胞水平上发生肾单位细胞死亡和修复以最小化细胞死亡并增强再生过程。斑马鱼背景是一个很好的模型系统来完成这个目标，因为它包含类似于哺乳动物肾单位的解剖段。以前，用于研究鱼类肾损伤的最常用模型是药物庆大霉素模型。然而，该模型不允许精确的时空控制损伤，因此难以研究涉及肾脏修复的细胞和分子过程。为了克服这个限制，这项工作提出了一种方法，与庆大霉素方法相反，特定的绿色荧光蛋白（GFP）-ex可以使用紫外激光（405nm）照射按压肾单位片段。这种AKI的新型模型提供了其他上皮损伤方法缺乏的许多优点。其主要优点是在健壮的体内动物模型中"拨"伤害水平和精确的时空控制能力。这种新方法有可能显着提高对肾损伤和修复机制的理解水平。

请注意，该协议已成功应用于许多肾转基因品系，包括ET（krt8:EGFP）sqet11-9，ET（krt8:EGFP）sqet33-d10和Tg（atp1a1a.4:GFP）。这里显示的示例结果是使用ET（krt8:EGFP）sqet11-9线获得的。 图1显示了实例的photoablation协议。在感兴趣的区域内监测平均GFP强度（ 图1A和电影1 ）。示例?...

Watch this Scientific Journal Video about Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish at JoVE.com

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

这项工作提出了使用肾GFP转基因斑马鱼的节段性肾损伤的新的体内模型。该模型允许诱导肾上皮细胞的靶向消融以显示肾单位损伤和修复的细胞机制。

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

precise-cellular-ablation-approach-for-modeling-acute-kidney-injury

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.6K 次观看.  NYITCOM. 这项工作提出了使用肾GFP转基因斑马鱼的节段性肾损伤的新的体内模型。该模型允许诱导肾上皮细胞的靶向消融以显示肾单位损伤和修复的细胞机制。 

视频: Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

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 ...

生物学

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 ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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 ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成人斑马鱼中西斯铂诱导的急性肾损伤模型

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

医学

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

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 decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

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 ...

Intravenous Microinjection of Nephrotoxins into Zebrafish Embryo: A Technique to Deliver Nephrotoxic Agents into the Bloodstream of Zebrafish Embryo

Source: McKee, Robert A. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/54241" target="_blank">Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates the intravenous microinjection of nephrotoxins into the tail vein of a zebrafish embryo to study their chemical effects on renal function.

将肾毒素静脉显微注射到斑马鱼胚胎中:一种将肾毒性药物输送到斑马鱼胚胎血液中的技术

Source: McKee, Robert A. et al. Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury. J. Vis. Exp.&#160;(2016)
This video demonstrates the ...