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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Petri Dishes, 35 x 10mm</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 15mm</td>
			<td>Midwest Scientific</td>
			<td>910</td>
			<td>Procedural Usage: Step 1</td>
		</tr>
		<tr>
			<td>De-chorination 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 2 , 0.33 mM MgSO 4 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, and 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</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 ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

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

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

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

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

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