This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

注:伦理学声明:所有实验均按照用于研究使用动物马萨诸塞州总医院的指导方针进行。 1.事前准备<ol><li>确定有多少成年斑马鱼6-12个月大的伤害。计划伤害10-20％以上的鱼比是必要的。为了尽量减少可变性，使用年龄匹配的同胞的鱼饲养在同一个罐，使它们大致相同的尺寸，并具有更少的遗传变异。母鱼更容易注入相比公鱼，由于腹部的更大的容量。但是，男性和女性给予了类似的结果。 </li><li>当做实验首次，绘制剂量曲线，以确定适当庆大霉素剂量为鱼的特定菌株。在实验中使用之前测试每一批新的庆大霉素庆大霉素以来的纯度一批不同于一批。适当的剂量应通过评估expressi确定上的损伤标记物如lhx1a。 （参见图1A - D）。 </li><li>准备庆大霉素解决方案。庆大霉素原液可被存储在-20℃，解冻后使用。例如，对于一个80毫克/公斤的剂量（80毫克庆大霉素/ kg体重的）在一个鱼体重0.5克，制备庆大霉素2毫克/毫升在磷酸盐缓冲盐水作为一种方便的工作溶液。这提供了一个20微升庆大霉素为腹腔注射成鱼。 注:在一般情况下，80-120毫克/千克取得良好的效果，但剂量低至40毫克/公斤，可适当。庆大霉素也可在溶液中购买，以尽量减少有害暴露于实验室人员。 注:庆大霉素高剂量可能是有毒的。戴上手套，重粉面膜的时候。 </li><li>制备100毫升0.016％三卡因水用于麻醉鱼。制备一个25倍的库存三卡因（4克/ 1L）溶于无菌milliQ水中的调节至pH 7，并储存在4℃直至使用。淡化4米升在100毫升鱼水的0.016％工作浓度。 注:三卡因是一种麻醉剂和皮肤有刺激性，搬运时戴上手套。 </li><li>做鱼舀出塑料移液管的切割灯泡成勺形和切割2槽底部排水。负载1毫升注射器（带10微升等级）与庆大霉素准备解决方案，并附加一个30½摹针。去除气泡。拧上的注射器针头，以确保在针的斜角尖背向和针筒标记是朝前和可读性。对于控制注射，准备一个注射器和针头用无菌PBS。 </li><li>准备一个规模用干净的表面或船的重量，以及纸巾晒鱼和鱼保持注射。 </li><li>准备个人的小半升有盖的容器举行的鱼一夜之间观察注射后。小透明塑料笼交配是用于此目的的。这些容器不应白色 - 理想，他们应该清楚，这样的鱼棚白色上皮管型在黑色的台面进行观察。填写每个容器有足够的鱼水的鱼游泳舒适。 </li></ol>庆大霉素2.腹腔注射<ol><li>在麻醉鱼水0.016％三卡因溶液的鱼。等待鳃通风量，以减缓和鱼不再响应触摸。 </li><li>舀起使用鱼舀鱼，从头部向尾部，以避免伤及鳃鳍或移动。放在纸巾鱼通过在其一侧轻轻转动捞出来吸收过量的水，从勺甩掉多余的水份。 </li><li>再舀上来的鱼，并把它在权衡船在归零规模和权衡鱼。一轮以最接近0.25克，并计算庆大霉素注射适量。 注:0.5g的鱼用20微升注射80毫克/公斤的剂量。 </li><li>再舀上来的鱼，并把它放在一个干燥的折叠纸巾。如果用右手注入，按住纸巾在左手，并把鱼的头指向左边，用肚皮方便。 </li><li>握住针注射器以45°角，腹部皮肤，先于泄殖腔。按刚下皮肤进针，然后下降的角度和向前滑动针在皮肤下，避免内脏。压低柱塞适量，暂停，以确保没有液体出来时分针，然后拔出针头。如果拿着鱼稳定是一个问题，对躯干肘部支撑，以稳定他们。 注:当处理庆大霉素溶液戴上手套。 </li><li>每条鱼落入一个单独的容器中。观察鱼，并保证从麻醉中复苏。保持鱼在28.5℃下过夜。 注:如果鱼没有立即恢复，使用反胶FER吸管灌溉水在其鳃来恢复它。 </li><li>当用相同剂量庆大霉素注入多个鱼使用相同的注射器和针头。处理用过的注射器和针头插入相应的生物危害锐器容器。 </li></ol>损伤3.注射后观察<ol><li>第二天（1天伤后），放置在其容器注入的鱼放在一个黑暗的表面。死亡的上皮组织中受伤的鱼排出的白色塑像应该是可见的。 （参见图1E - H）。如果没有管型，无论是鱼由庆大霉素注射没有受伤（或者剂量太低，或者一些在注射过程中泄漏的庆大霉素）或鱼严重受伤导致输尿管和泄殖腔的完全阻断与脱落的组织。 注:如果鱼是无法清除其体内的塑像它通常会在2-3天内死亡，因此不能用于再测定。如果没有强制转换是可见的，通过浸渍至少10分钟鳃运动停止后安乐死在三卡因水中的鱼。 </li><li>如果白石膏组织可见，抛开鱼并继续检查其他鱼类。庆大霉素的适当的剂量将导致鱼是可用的80-90％。池鱼受伤，然后将它们如果需要分成不同的治疗组。 </li></ol> 4.护理恢复的鱼<ol><li>保持鱼在清水和拥挤的环境。脏水和拥挤会导致感染和意外死亡。如果可能，尝试每天保持不超过6尾/500毫升鱼水和换水。 注意:这是一个最小的体积为节约空间或如果研究者希望执行使用昂贵的药物治疗实验。 </li><li>不要喂的鱼，直到后3天的伤害。恢复鱼不会吃第一，并在坦克任何食品会发生分解，并促进细菌克owth。第3天开始伤后，每天喂一次少量。 </li></ol>肾脏损伤5.分析<ol><li>对于为 lhx1a表达原位杂交，肾脏可以从鱼都收获希望的时间点。冰在三卡因水中鱼实施安乐死，至少10分钟，直至鳃运动停止，然后取出头用刀片并打开体腔，并用钳子取出内脏。 </li><li>留在原地（贴在背体壁色素器官），肾和PBS固定在4％多聚甲醛鱼过夜。解剖出肾15并继续原位杂交标准。 <ol><li>简要地说，洗净肾脏在PBST（磷酸盐缓冲盐水0.5％吐温20），进行1小时，在室温下透化用蛋白酶K在PBST（10微克/毫升）。用4％多聚甲醛后固定，并用PBST再洗涤。 </li><li>然后预杂交肾脏过夜68;℃下在杂交缓冲液（50％甲酰胺，5×SSC，50微克/毫升肝素，500微克/毫升的tRNA，0.1％吐温20，pH 6.0）中。 </li><li>孵育在杂交缓冲液中地高辛标记探针的样品中，然后再洗涤5-10分钟每次用100％，75％，50％，25％，杂交缓冲液在68℃，然后转移到室温，并清洗两次，每次30分钟，用2×SSC，0.1％吐温20，和清洗两次，每次30分钟，用0.​​2×SSC，0.1％吐温20。 </li><li>平衡样品3×5分钟，在生物圈（0.1M马来酸，0.15M氯化钠，pH值7.5）和封闭过夜，在4℃在生物圈用10％山羊血清。然后孵育样本过夜抗地高辛-AP的Fab 1:5000人与生物圈。 </li><li>洗样品5倍1小时的人与生物圈计划，并平衡3倍于NTMT 15分钟（0.1米的Tris pH9.5，0.05M的氯化镁 ，0.01 M氯化钠，50微升吐温20）。 </li><li>然后对待NBT / BCIP肾脏检测信号。固定用4％多聚甲醛的肾脏并孵育在二甲基甲酰胺字符型数据已经超额NBT / BCIP，在去离子水中漂白过夜。洗肾脏，然后拍摄在PBST中与下一个盖玻片50％甘油。 </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

斑马鱼是理想的学习成人器官再生包括成人中肾肾脏6。最近的研究已经利用分子标记技术和转基因新记者行更好地表征在肾再生发生的步骤，哪些细胞可能是负责7,12。尿管型的观察已使用了一个多世纪来诊断肾脏疾病在人类16。在这里，我们使用管型的存在，作为庆大霉素注射后一种简单，非侵入性的和早期指标的急性肾损伤的程度。我们观察到的鱼，不产生强制类型转...

肾脏是流体平衡，血压调节和过滤废物从体内必需的。虽然哺乳动物可以利用受伤分化的上皮细胞后1-4修复肾单位存在，他们似乎缺乏保留的干细胞5的游泳池，并不能形成新的肾单位从头 。在对比哺乳动物，成鱼能够形成整个成年生活新肾支持鱼的生长，并且响应于损伤6,7。斑马鱼， 斑马鱼 ，是一种宝贵的模式生物器官再生8-10的研究，并有可能提供强大的洞察到应用程序中来改造人类的肾脏的修复。 的Tg（Lhx1a:EGFP）转基因11已经显示然而标记肾祖细胞的池，在成年斑马鱼肾12，控制lhx1a的响应+细胞损伤ř机制emain不清楚。 氨基糖甙类的庆大霉素是一种广泛使用抗生素与人类已知的13和肾耳毒性作用。腹腔注射庆大霉素是一家成立于鱼6诱发急性肾损伤的方法。这种损伤的鱼模仿的肾小管上皮细胞，而发生在人类过量服用庆大霉素后14肾小球疤痕的损失。斑马鱼中的庆大霉素注射诱导损伤是引起强烈的，同步再生反应，产生了许多新的肾单位，同时继续通过形成，增殖和分化阶段的便捷方式。 该协议通过利用无创可视化的筛选过程中尽量减少离群点细节我们的方法可再生的损伤的成年斑马鱼肾脏。我们注意到，与庆大霉素损伤导致的肾上皮组织和格式死亡的事实优势离子肾小管演员，然后积聚成群众中肾管和泄殖腔。这些是由鱼传递，并且可以在水目视观察。这使我们能够对鱼类具有较强的非致命伤害，然后可以为汇集进一步的实验筛选。最小化的未受伤的鱼或鱼，才到达实验的终点导致更均匀和高效的数据收集和分析该模具的数量。此外，没有任何特殊的设备或试剂要求，使得这种方法具有成本效益，并适用于在学术或教学环境中使用。使用我们的方法，在这里，我们表明肾再生庆大霉素剂量的增加效果以及增加温度的作用。

<table><tbody><tr><td>gentamicin sodium sulfate</td><td>Sigma</td><td>G1264</td><td>TOXIC, purity varies from batch to batch</td></tr><tr><td>plastic transfer pipets</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>1 ml Norm-Ject syringes</td><td>Electron Microscopy Sciences</td><td>72520</td><td>green plastic syringes, ordinary 1 ml syringes are OK, but harder to read accurately</td></tr><tr><td>30 G1/2 needles</td><td>Becton Dickinson</td><td>305106</td><td></td></tr><tr><td>ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td><td>Sigma</td><td>A5040</td><td>IRRITANT</td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Make 4% in 1x PBS for working solution</td></tr></tbody></table>

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.

庆大霉素损伤可目视由肾上皮管型的观察中的水（ 图1）的确认。庆大霉素不同剂量被用来伤害野生成年斑马鱼TuAB，导致在增加再生肾脏lhx1a +细胞聚集（ 图1A - D）的数字。白石膏可以很容易地看出，在水下1伤天之后（ 图1E - H）。庆大霉素低剂量导致不具有强制类型转换大多数鱼，而高剂量导致了鱼类有管型（ 图1I）</s...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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 ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Research

JoVE Journal

Developmental Biology

10.8K 次观看.  Massachusetts General Hospital. 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. 

视频: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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

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 adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

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 ...

科研

发育生物学

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 ...

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

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

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 ...

神经科学

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

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 ...

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 ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

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 ...

肾再生的成年斑马鱼庆大霉素性损伤

摘要

探索更多视频

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

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

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

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

直接进入成年斑马鱼的肾细胞移植

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

对于侧线再生成年斑马鱼的含量

肾单位的组成与功能的成年斑马鱼肾脏分析

成人斑马鱼中西斯铂诱导的急性肾损伤模型

肾再生的成年斑马鱼庆大霉素性损伤

摘要

探索更多视频

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

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

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

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

直接进入成年斑马鱼的肾细胞移植

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

对于侧线再生成年斑马鱼的含量

肾单位的组成与功能的成年斑马鱼肾脏分析

成人斑马鱼中西斯铂诱导的急性肾损伤模型

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