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 border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin sodium sulfate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">G1264</td>
			<td>TOXIC, purity varies from batch to batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic transfer pipets</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 ml Norm-Ject syringes</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">72520</td>
			<td>green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30G1/2 needles</td>
			<td style="width:101px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>IRRITANT</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">16% paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td>Make 4% in 1xPBS 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

Research

JoVE Journal

Developmental Biology

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

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.

Lesão Vessel Mecânica em embriões Zebrafish

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

Biologia do Desenvolvimento

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.

Ablação específicas do hepatócito em Zebrafish para estudar a regeneração do fígado-driven biliar

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.

Técnica ao Target microinjeção para o desenvolvimento<em&gt; Xenopus</em&gt; 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 ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Desenvolvimento de um modelo de fígado fibrótica induzida por etanol no peixe-zebra para estudar Progenitor mediada por células de regeneração de hepatócitos

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Cultura do Adulto Transgênicos Zebrafish Retinal explantes for Imaging Em células vivas, por Multiphoton Microscopia

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

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 ...

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.

Análises histológicas de lesão hepática alcoólica aguda em peixe-zebra

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 ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Um método simplificado e eficiente para isolar os queratinócitos humanos primários do tecido da pele de adultos

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

SiRNA Nanoparticle-mediada-silenciamento no coração de Zebrafish adulto

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

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.

Modelos de lesão adulto Zebrafish para estudar os efeitos de prednisolona na regeneração de tecido ósseo

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