Name: kidney regeneration in adult zebrafish by gentamicin induced injury
Uploaded: 2015-08-03T14:30:00.000+00:00
Duration: 6 min 25 s
Description: Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

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.

Declaração de Ética: NOTA: Todos os experimentos foram realizados de acordo com orientações Hospital Geral de Massachusetts para o uso de animais em pesquisa. 1. Preparação Prévia <ol><li> Determine quantas adulto zebrafish 6-12 meses de idade para ferir. Plano de ferir 10-20% mais peixes do que será necessário. A fim de minimizar a variabilidade, o uso combinado de peixe irmão idade criados em conjunto no mesmo tanque de modo que eles são aproximadamente do mesmo tamanho e têm menor variabilidade genética. Peixe fêmea são muito mais fáceis de injectar em comparação com o macho de peixe, devido à maior capacidade do abdómen. No entanto, os machos e as fêmeas dão resultados semelhantes. </li><li> Ao fazer a experiência, pela primeira vez, traçar uma curva de dose para determinar a dosagem apropriada de gentamicina para uma estirpe específica de peixe. Testar cada novo lote de gentamicina antes do uso em experiências uma vez que a pureza de gentamicina varia de lote para lote. Dosagem adequada deve ser determinada pela avaliação expressisobre marcadores de prejuízo, tais como lhx1a. (Ver Figura 1A - D). </li><li> Preparar a solução de gentamicina. Solução de estoque de gentamicina pode ser armazenado em -20 ° C e descongelada para utilização posterior. Por exemplo, para um 80 mg / kg (80 mg gentamicina / kg de peso corporal) num peixe pesando 0,5 g, preparar a 2 mg / ml de gentamicina em solução salina tamponada com fosfato como uma solução de trabalho conveniente. Isto proporciona uma 20 uL de gentamicina por injecção intraperitoneal no peixe. NOTA: De um modo geral, 80-120 mg / kg de alcançar bons resultados, mas doses tão baixas quanto 40 mg / kg podem ser adequadas. Gentamicina também podem ser comprados em solução para minimizar a exposição perigosos para o pessoal de laboratório. NOTA: Gentamicina em doses elevadas pode ser tóxico. Usar luvas e máscara quando pesando pó. </li><li> Prepare 100 ml de 0,016% tricaina água para anestesiar peixes. Preparar um estoque de tricaina (4 g / 1 L) dissolvido em água MilliQ estéril de 25x ajustar até pH 7 e armazenar a 4 ° C até à sua utilização. Diluir 4 ml em 100 ml de água de peixe para uma concentração de trabalho de 0,016%. NOTA: tricaina é um anestésico e pele irritante, usar luvas ao manusear. </li><li> Faça um peixe colher fora de uma pipeta de transferência de plástico cortando o bulbo em forma de colher e corte de 2 slots no fundo para drenar a água. Seringas de 1ml de carga (com 10 gradações ul) com a solução de gentamicina preparado e anexar uma agulha G 30½. Remova as bolhas de ar. Torça a agulha da seringa para se certificar de que a ponta angular sobre a agulha está enfrentando afastado e as marcas de seringa são voltadas para a frente e legível. Para injeções de controle, preparar uma seringa e agulha estéril com PBS. </li><li> Prepare uma escala com uma superfície limpa ou pesar barco, bem como toalhas de papel para secar peixes e para a realização de peixes para injecção. </li><li> Prepare pequenos recipientes individuais de meio litro com tampa para a realização de peixes para observação pós durante a noite injeção. As pequenas gaiolas transparentes acasalamento plástico são úteis para este fim. EstesOs contentores não deve ser branco - idealmente, devem ser claras para que os moldes epiteliais brancas derramadas pelos peixes podem ser observados numa bancada de preto. Encha cada recipiente com água peixe suficiente para os peixes a nadar confortavelmente. </li></ol> 2. Injecção Intraperitoneal de gentamicina <ol><li> Anestesiar os peixes em solução tricaina 0,016% em água peixe. Aguarde até que a taxa de ventilação branquial a abrandar e que o peixe já não respondem ao toque. </li><li> Recolher o peixe usando a colher peixes, movendo-se de cabeça na direção da cauda, ​​a fim de evitar ferir as guelras ou barbatanas. Coloque o peixe em papel toalha para absorver o excesso de água, girando o peixe fora suavemente em seu lado e sacuda o excesso de água do furo. </li><li> Recolher o peixe novamente e colocá-lo no barco pesar na escala zerada e pesar o peixe. Redondo com a aproximação de 0,25 g, e calcular a quantidade adequada de gentamicina a injectar. NOTA: Para uma 0,5 g de peixe usam uma injecção de 20 ul às 80 mg / kg dose. </li><li> Recolher o peixe novamente e coloque-o sobre uma toalha de papel dobrado seco. Se injetar com a mão direita, segure a toalha de papel na mão esquerda e coloque a cabeça do peixe apontando para a esquerda, com a barriga de fácil acesso. </li><li> Segurar a seringa com a agulha com um ângulo de 45 ° para a pele do ventre, anterior para a cloaca. Empurrar a agulha debaixo da pele, então diminuir o ângulo e deslizar para a frente a agulha sob a pele, evitando os órgãos internos. Empurrar o êmbolo a quantidade adequada, pausa para garantir que nenhum líquido está saindo em torno da agulha, em seguida, retirar a agulha. Se segurando o peixe é um problema constante, cotovelos cinta contra o torso para estabilizá-los. NOTA: Utilize luvas ao manusear solução de gentamicina. </li><li> Solte cada peixe em um recipiente individual. Observar os peixes e garantir a sua recuperação da anestesia. Manter os peixes a 28,5 ° C durante a noite. NOTA: Se o peixe não reviver imediatamente, use um plástico transfer pipeta para irrigar água através de suas brânquias de reanimá-lo. </li><li> Utilizar a mesma seringa e agulha, aquando da injecção peixe múltiplos com a mesma dose de gentamicina. Elimine a seringa ea agulha usado para o recipiente de risco biológico farelos apropriado. </li></ol> 3. Publicar Injeção Observação de Lesão <ol><li> No dia seguinte (um dia após a lesão), coloque o peixe injetado em seu recipiente sobre uma superfície escura. Elencos brancas de tecido epitelial mortos excretado pelos peixes feridos devem ser visíveis. (Ver Figura 1E - H). Se não existem moldes, ou o peixe não foi lesionado por injecção de gentamicina (ou a dose é muito baixa, ou alguma da gentamicina vazado durante o processo de injecção) ou o peixe foi gravemente ferido, resultando em um bloqueio completo dos ureteres e da cloaca com tecido descartado. NOTA: Se o peixe é incapaz de limpar os moldes de seu corpo, geralmente morrem dentro de 2-3 dias e é, portanto, inutilizáveis ​​por mais tempoensaios. Se não há elencos são visíveis, eutanásia do peixe na água tricaina por imersão durante pelo menos 10 minutos após o movimento de emalhar pára. </li><li> Se elencos dos tecidos brancos são visíveis, defina o peixe de lado e continuar a verificar os outros peixes. Uma dose apropriada de gentamicina resultará em 80-90% de o peixe ser utilizável. A piscina do peixe ferido e, em seguida, divida-os em diferentes grupos de tratamento, se desejar. </li></ol> 4. Cuidados de Recuperação de Peixe <ol><li> Manter os peixes em água limpa e meio ambiente sem aglomeração. A água suja e aglomeração vai levar a infecções e morte não intencional. Tente manter não mais do que 6 peixes / 500 ml de água de peixe e trocar a água diariamente, se possível. NOTA: Este é um volume mínimo para a conservação do espaço ou se o investigador deseja realizar experimentos usando tratamentos com medicamentos caros. </li><li> Não alimente o peixe até 3 dias após a lesão. Recuperando peixe não vai comer no início, e qualquer alimento no tanque vai se decompor e promover bactérias growth. A partir de 3 dias após o ferimento, alimentar uma pequena quantidade, uma vez por dia. </li></ol> 5. Análise de feridos Rins <ol><li> Para a hibridização in situ para a expressão do mRNA lhx1a, rins podem ser colhidas a partir de peixes em intervalo de tempo desejados. Euthanize peixe na água tricaina em gelo pelo menos 10 minutos até que o movimento de emalhar pára, em seguida, remover a cabeça com uma lâmina de barbear e abrir a cavidade do corpo e remover os órgãos internos com uma pinça. </li><li> Deixar o rim no lugar (órgão pigmentada fixada na parede do corpo dorsal) e corrigir o peixe em paraformaldeído a 4% em PBS durante a noite. Dissecar o rim 15 e continuar com o padrão de hibridação in situ. <ol><li> Resumidamente, lavar os rins em PBST (solução salina tamponada com fosfato 0,5% de Tween 20), permeabilizadas com proteinase K em PBST (10 ug / ml) durante 1 hora à temperatura ambiente. Pós-corrigir com paraformaldeído a 4%, e lavou-se novamente com PBST. </li><li> Em seguida, pré-hibridação dos rins durante a noite a 68; ° C em tampão de hibridação (formamida a 50%, 5x SSC, 50 ug / ml de heparina, 500 ug / ml de ARNt, 0,1% de Tween 20, pH 6,0). </li><li> Incubar as amostras com digoxigenina marcado sonda em tampão de hibridação, em seguida, lavar durante 5-10 minutos cada, com 100%, 75%, 50%, 25% de tampão de hibridação a 68 ° C, em seguida, mudou-se para a temperatura ambiente e lava-se duas vezes durante 30 min com 2 x SSC com 0,1% de Tween 20, e lava-se duas vezes durante 30 min com 0,2x SSC com 0,1% de Tween20. </li><li> Equilibrar a amostra 3x 5 min em MAB (0,1 M de ácido maleico, 0,15 M de NaCl, pH 7,5) e bloqueadas durante a noite a 4 ° C em MAB com 10% de soro de cabra. Em seguida, incubar amostras durante a noite em anti-digoxigenina-AP Fab 1: 5.000 em MAB. </li><li> Lava-se a 5x amostra durante 1 h em MAB, e equilibrar 3x durante 15 minutos em NTMT (0,1 M de Tris pH 9,5, 0,05 M de MgCl2, 0,01 M de NaCl, 50 uL de Tween 20). </li><li> Em seguida, tratar os rins com NBT / BCIP para detectar o sinal. Corrigir os rins com paraformaldeído a 4% e incubadas em dimetilformamida a remove excesso de NBT / BCIP, branqueada durante a noite em água deionizada. Lavar os rins e, em seguida, fotografar em PBST com 50% de glicerol sob uma lamela. </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

O peixe-zebra é ideal para estudar a regeneração de órgãos adultos, incluindo o rim adulto mesonephric 6. Estudos recentes têm se aproveitado de marcadores moleculares e novas linhas repórter transgénico para melhor caracterizar as etapas que ocorrem durante a regeneração de néfrons e quais as células podem ser responsáveis ​​7,12. Observação de cilindros urinários foi usado para mais de um século, para diagnosticar a doença renal em seres humanos 16. Aqui usamos a ...

O rim é essencial para a homeostase dos fluidos, regulação da pressão arterial e filtragem de resíduos do corpo. Embora os mamíferos são capazes de reparar néfrons existentes após a lesão usando células epiteliais diferenciadas 1-4, eles parecem não ter um pool de células-tronco reservadas 5 e são incapazes de formar novos néfrons de novo. Em contraste com os mamíferos, peixes adultos são capazes de formar novas nefrónios ao longo da vida adulta para apoiar o crescimento dos peixes e, em resposta a uma lesão 6,7. O peixe-zebra, Danio rerio, é um organismo modelo de valor inestimável para o estudo da regeneração de órgãos 10/08 e tem o potencial de fornecer insights poderosos em aplicações de engenharia a reparação de rins humanos. A Tg (Lhx1a: EGFP) transgene 11 foi mostrado para identificar um conjunto de células progenitoras no nefrónio do rim adulto zebrafish 12, no entanto, os mecanismos que controlam a resposta de células lhx1a + para r lesãoemain claro. A gentamicina é um antibiótico aminoglicosídeo amplamente utilizado com efeitos nefrotóxicos e ototóxicos conhecidos em humanos 13. Injeção intra-peritoneal de gentamicina é um método estabelecido de induzir lesão renal aguda em peixes 6. Esta lesão em peixes imita a perda de epitélio tubular e cicatrizes de glomérulos que ocorre em seres humanos após overdose de gentamicina 14. Indução de lesão no peixe-zebra por injecção de gentamicina é uma maneira conveniente de induzir uma resposta forte de regeneração, síncrona, com muitas novas nefrónios produzidos e procedendo em simultâneo através de estágios de formação, diferenciação e proliferação. Este protocolo detalhes o nosso método de lesão robusta e reproduzível no peixe-zebra rim adulto, utilizando um processo de triagem visual não invasivo para minimizar valores atípicos. Nós tirar proveito do facto de que a lesão com gentamicina leva à morte do tecido renal epitelial e formatoion de moldes tubulares renais, que depois se acumulam em massas nas condutas mesonéfricos e cloaca. Estes são passados ​​pelos peixes e pode ser observada visualmente na água. Isto permite-nos para triagem de peixe com forte lesão não-letal, que pode então ser combinadas para fazer novas experiências. Minimizando o número de peixes ou peixe ileso que morrem antes de chegar ao ponto final do experimento leva a mais uniforme e eficiente de coleta de dados e análise. Além disso, não são necessários dispositivos especiais ou reagentes, tornando este método custo-efetivo e adequado para uso em um ambiente acadêmico ou de ensino. Utilizando o nosso método, aqui mostram os efeitos de doses crescentes de gentamicina sobre a regeneração do rim, assim como o efeito do aumento da temperatura.

<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.

Lesão de gentamicina pode ser confirmado visualmente pela observação de moldes epiteliais renais em água (Figura 1). Diferentes doses de gentamicina foram usadas para ferir adulto zebrafish wildtype TuAB, resultando em um número crescente de lhx1a + agregados celulares no rim regeneração (Figura 1A - D). Gesso branco pode ser facilmente visto na água um dia após a lesão (Figura 1E - H). Uma baixa dose de gentamicina r...

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.

A regeneração renal em Zebrafish Adulto por lesão induzida pela gentamicina

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

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10.8K visualizações.  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. 

Vídeo: 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.

Abordagem precisa de ablação celular para modelagem de lesão renal aguda no desenvolvimento de peixe-zebra

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 ...

Biologia do Desenvolvimento

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.

Stab ferida ferimento do peixe-zebra adulto Telencéfalo: Um método para investigar Vertebrados Cérebro Neurogênese e Regeneração

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

Neurociência

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.

Microinjeções intravenosa de larvas do peixe para o Estudo de lesão renal aguda

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

Biologia

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.

Modelo de Lesão por Arma Branca do Adulto Óptico Utilizando Zebrafish e Medaka para Análise Comparativa da Capacidade Regenerativa

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.

Transplante de células diretamente no Rim de Adulto 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 ...

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.

Dissecção do rim Zebrafish Adulto

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 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.

Ablação a laser da Pronephros Zebrafish para estudo da regeneração epitelial renal

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.

Um ensaio para a lateral de Regeneração de Linha em Zebrafish Adulto

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.

Análise de Composição Nephron e função no rim adulto Zebrafish

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.

Modelo de lesão renal aguda induzida por cisplatina em zebrafish adulto

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