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.

NOTA: Etica Dichiarazione: Tutti gli esperimenti sono stati condotti in conformità alle linee guida del Massachusetts General Hospital per l&#39;impiego di animali nella ricerca. 1. preparazione preliminare <ol><li> Determinare il numero di adulti zebrafish 6-12 mesi di ferire. Piano per ferire il 10-20% in più pesce di quanto sarà necessario. Per ridurre al minimo la variabilità, utilizzo età abbinato pesci allevati sibling insieme nella stessa vasca in modo che siano all&#39;incirca le stesse dimensioni e la variabilità genetica meno. Pesci femminili sono molto più facile per iniettare rispetto al pesce maschio, a causa della maggiore capacità dell&#39;addome. Tuttavia, i maschi e le femmine danno risultati simili. </li><li> Quando si esegue l&#39;esperimento per la prima volta, tracciare una curva dose per determinare il dosaggio appropriato gentamicina per uno specifico ceppo di pesci. Verificare ogni nuovo lotto di gentamicina prima dell&#39;uso in esperimenti dal momento che la purezza di gentamicina varia da lotto a lotto. La dose deve essere determinato valutando expressisu di marcatori di pregiudizio, come lhx1a. (Vedi Figura 1A - D). </li><li> Preparare la soluzione di gentamicina. Gentamicina soluzione madre può essere conservata a -20 ° C e scongelato per un uso successivo. Ad esempio, per una dose di 80 mg / kg (80 mg / kg gentamicina di peso corporeo) in un pesce del peso di 0,5 g, preparare 2 mg / ml di gentamicina in tampone fosfato come una soluzione di lavoro comoda. Questo fornisce un 20 microlitri di gentamicina per iniezione intraperitoneale nel pesce. NOTA: In generale, 80-120 mg / kg ottenere buoni risultati, ma dosi di 40 mg / kg può essere appropriato. Gentamicina può anche essere acquistato in soluzione per ridurre al minimo l&#39;esposizione pericolosa al personale di laboratorio. NOTA: Gentamicina in dosi elevate può essere tossico. Indossare guanti e maschera nel valutare in polvere. </li><li> Preparare 100 ml di acqua tricaine 0,016% per anestetizzare il pesce. Preparare uno stock 25x di tricaine (4 g / 1 L) disciolto in acqua sterile MilliQ regolare a pH 7 e conservare a 4 ° C fino al momento dell&#39;uso. Diluire 4 ml in 100 ml di acqua pesce per la soluzione di lavoro di 0,016%. NOTA: Tricaine è un anestetico e pelle irritante, indossare guanti durante la manipolazione. </li><li> Fare un pesce paletta da un pipetta plastica tagliando la lampadina a forma di paletta e taglio 2 slot nella parte inferiore per drenare l&#39;acqua. Siringhe carico 1ml (con 10 gradazioni microlitri) con la soluzione di gentamicina preparata e attribuiscono un ago G 30½. Rimuovere eventuali bolle d&#39;aria. Girare l&#39;ago della siringa per fare in modo che la punta angolata sul ferro sia rivolta e le marcature siringa si trovano ad affrontare in avanti e leggibile. Per le iniezioni di controllo, preparare una siringa e l&#39;ago con PBS sterile. </li><li> Preparare una scala con una superficie pulita o pesare barca così come i tovaglioli di carta per asciugare il pesce e per lo svolgimento di pesce per l&#39;iniezione. </li><li> Preparare singoli contenitori piccole da mezzo litro con coperchio per lo svolgimento di pesce per l&#39;iniezione di osservazione posto durante la notte. Piccole gabbie di plastica accoppiamento trasparenti sono utili per questo scopo. Questecontenitori non devono essere bianco - idealmente dovrebbero essere chiaro in modo che i calchi epiteliali bianchi versate dai pesci possono essere osservati in cima a una panchina nera. Riempire ogni contenitore con acqua abbastanza pesce per il pesce a nuotare comodamente. </li></ol> 2. iniezione intraperitoneale di gentamicina <ol><li> Anestetizzare il pesce nella soluzione tricaine 0,016% in acqua i pesci. Attendere che il tasso di ventilazione branchia per rallentare e per il pesce a rispondere non più al tatto. </li><li> Raccogliere il pesce con la paletta pesce, muovendo dalla testa verso la coda, al fine di evitare di ferire le branchie o pinne. Mettere il pesce su carta assorbente per assorbire l&#39;acqua in eccesso ruotando il pesce con delicatezza su un fianco e scrollarsi di dosso l&#39;acqua in eccesso dal scoop. </li><li> Raccogliere il pesce di nuovo e metterlo in barca pesare sulla bilancia azzerato e pesare il pesce. Rotonda con l&#39;approssimazione di 0,25 g, e calcolare la giusta quantità di gentamicina da iniettare. NOTA: Per un 0,5 g di pesce utilizzare una iniezione di 20 ml a 80 dose mg / kg. </li><li> Raccogliere il pesce di nuovo e metterlo su un tovagliolo di carta piegato a secco. Se l&#39;iniezione con la mano destra, tenere il tovagliolo di carta nella mano sinistra e mettere la testa del pesce che punta a sinistra, con la pancia facilmente accessibile. </li><li> Tenere la siringa con l&#39;ago con un angolo per la pelle della pancia, anteriore alla cloaca 45 °. Spingere l&#39;ago sotto la pelle, quindi diminuire l&#39;angolo e far scorrere l&#39;ago in avanti sotto la pelle, evitando gli organi interni. Spingere il pistone la giusta quantità, mettere in pausa per assicurarsi che nessun liquido esce intorno all&#39;ago, quindi estrarre l&#39;ago. Se tiene il pesce costante è un problema, gomiti bretelle contro tronco per essere stabilizzati. NOTA: Maneggiare con guanti soluzione di gentamicina. </li><li> Eliminare ogni pesce in un singolo contenitore. Osservare il pesce e assicurarne il recupero dall&#39;anestesia. Mantenere pesce a 28,5 ° C durante la notte. NOTA: Se il pesce non rivivere subito, utilizzare un trans plasticafer pipetta per irrigare l&#39;acqua attraverso le branchie di rianimarlo. </li><li> Utilizzare lo stesso siringa e l&#39;ago quando si inietta pesce multipla con la stessa dose di gentamicina. Smaltire la siringa usata e l&#39;ago nel apposito contenitore per rischio biologico taglienti. </li></ol> 3. osservatorio Iniezione di lesioni <ol><li> Il giorno successivo (lesione dopo 1 giorno), posizionare il pesce iniettato nel suo contenitore su una superficie scura. Calchi bianche di tessuto epiteliale morti escreta dai pesci feriti dovrebbero essere visibili. (Vedi Figura 1E - H). Se non ci sono calchi, o il pesce non è stato ferito dall&#39;iniezione gentamicina (sia la dose era troppo bassa, o alcune delle gentamicina fuoriuscito durante il processo di iniezione) o il pesce era gravemente ferito conseguente blocco completo degli ureteri e cloaca con il tessuto sloughed. NOTA: Se il pesce non è in grado di cancellare i calchi dal suo corpo che di solito muoiono entro 2-3 giorni ed è quindi inutilizzabile per piùsaggi. Se non calchi sono visibili, eutanasia il pesce in acqua tricaine immergendo per almeno 10 minuti dopo il movimento branchia si arresta. </li><li> Se calchi tessuto bianche sono visibili, impostare il pesce da parte e continuare a controllare lo altri pesci. Una dose appropriata di gentamicina comporterà 80-90% del pesce essere utilizzabile. Pool i pesci feriti e poi divisi in diversi gruppi di trattamento, se desiderato. </li></ol> 4. Cura di Recupero di pesce <ol><li> Tenere pesce in acqua pulita e ambiente poco affollate. Acqua sporca e affollamento sarà portare a infezioni e la morte involontaria. Cercate di mantenere non più di 6 pesci / 500 ml di acqua pesce e cambiare l&#39;acqua ogni giorno, se possibile. NOTA: Si tratta di un volume minimo per la conservazione di spazio o se il ricercatore desidera effettuare esperimenti utilizzando trattamenti farmacologici costosi. </li><li> Non date da mangiare il pesce fino a 3 giorni dopo l&#39;infortunio. Pesce Recupero non mangeranno in un primo momento, e qualsiasi cibo nel serbatoio si decompongono e promuovere batteri growth. Partendo a 3 giorni dopo lesioni, alimentare una piccola quantità una volta al giorno. </li></ol> 5. Analisi di feriti Reni <ol><li> Per l&#39;ibridazione in situ per l&#39;espressione lhx1a mRNA, i reni possono essere raccolte da pesce timepoints desiderati. Eutanasia pesce in acqua tricaine su ghiaccio di almeno 10 minuti fino a quando il movimento branchia si arresta, quindi rimuovere la testa con una lametta e aprire la cavità del corpo e rimuovere gli organi interni con una pinza. </li><li> Lasciare il rene a posto (organo pigmentato attaccato alla parete dorsale del corpo) e fissare il pesce in 4% paraformaldeide in PBS durante la notte. Sezionare il rene 15 e continuare con lo standard ibridazione in situ. <ol><li> Brevemente, lavare i reni in PBST (tampone fosfato 0,5% Tween 20), permeabilizzate con proteinasi K in PBST (10 ug / ml) per 1 ora a temperatura ambiente. Post-fissare con 4% paraformaldeide, e lavato ancora con PBST. </li><li> Poi pre-ibridare i reni durante la notte a 68; ° C in tampone di ibridazione (50% formammide, 5x SSC, 50 ug / ml di eparina, 500 ug / ml tRNA, 0,1% Tween20, pH 6,0). </li><li> Incubare i campioni con digossigenina marcato sonda nel tampone di ibridazione, quindi lavare per 5-10 min ciascuno con 100%, 75%, 50%, 25% tampone di ibridazione a 68 ° C, poi si trasferisce a temperatura ambiente e lavata due volte per 30 min con 2x SSC con 0,1% Tween20, e lavare due volte per 30 min con 0.2x SSC con 0,1% di Tween20. </li><li> Equilibrare campione 3x 5 min a MAB (0,1 M di acido maleico, 0,15 M di NaCl, pH 7,5) e bloccate notte a 4 ° C in MAB con siero di capra 10%. Poi incubare i campioni durante la notte in anti-digossigenina-AP Fab 1: 5.000 a MAB. </li><li> Lavare il 5x campione per 1 ora in MAB ed equilibrare 3x per 15 min a NTMT (0.1 M Tris pH9.5, 0,05 M MgCl 2, 0,01 M di NaCl, 50 microlitri Tween20). </li><li> Poi trattare i reni con NBT / BCIP per rilevare il segnale. Fissare i reni con paraformaldeide al 4% e incubate in dimetilformammide a remove eccesso NBT / BCIP, sbiancato notte in acqua deionizzata. Lavare i reni e poi fotografare in PBST con il 50% di glicerolo sotto un vetrino. </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

Il pesce zebra è ideale per studiare la rigenerazione di organi adulti tra cui il rene mesonefrico 6 adulti. Recenti studi hanno approfittato di marcatori molecolari e nuove linee giornalista transgenici per caratterizzare meglio i passaggi che si verificano durante la rigenerazione nefrone e quali le cellule potrebbero essere responsabili 7,12. Osservazione di calchi urinari è stata utilizzata da più di un secolo per diagnosticare la malattia del rene nell&#39;uomo 16. Qui si usa la ...

Il rene è essenziale per l&#39;omeostasi del fluido, la regolazione della pressione sanguigna e la filtrazione dei rifiuti dal corpo. Anche se i mammiferi sono in grado di riparare i nefroni esistenti dopo la lesione utilizzando cellule epiteliali differenziate 1-4, sembrano mancare di un pool di cellule staminali riservati 5 e sono in grado di formare nuovi nefroni de novo. A differenza dei mammiferi, i pesci adulti sono in grado di formare nuovi nefroni per tutta la vita adulta per sostenere la crescita del pesce e in risposta al danno 6,7. Il zebrafish, Danio rerio, è un organismo modello prezioso per lo studio della rigenerazione degli organi 8-10 e ha il potenziale per fornire potenti intuizioni in applicazioni per l&#39;ingegneria la riparazione di reni umani. Il Tg (Lhx1a: EGFP) transgene 11 ha dimostrato di etichettare un pool di cellule progenitrici nephron in zebrafish adulti rene 12, tuttavia i meccanismi che controllano la risposta delle cellule lhx1a + al danno rEmain chiaro. La gentamicina aminoglicoside è un antibiotico ampiamente usato con noti effetti nefrotossici e ototossici nell&#39;uomo 13. Iniezione intraperitoneale di gentamicina è un metodo consolidato di indurre danno renale acuto nei pesci 6. Questa lesione nel pesce imita la perdita dell&#39;epitelio tubolare e cicatrici di glomeruli che si verifica negli esseri umani dopo gentamicina sovradosaggio 14. Indurre lesioni in zebrafish per iniezione gentamicina è un modo conveniente di indurre una forte risposta rigenerazione sincrono, con molti nuovi nefroni prodotte e, contemporaneamente, di procedere attraverso le fasi di formazione, la proliferazione e la differenziazione. Dettagli Questo protocollo il nostro metodo per lesioni robusto e riproducibile in zebrafish adulti rene utilizzando un processo di screening non invasivo visivo per ridurre al minimo i valori anomali. Approfittiamo del fatto che il pregiudizio con gentamicina porta alla morte del tessuto renale epiteliali e formatoioni di calchi tubulari renali, che poi si accumulano in masse nei condotti mesonefrici e cloaca. Questi sono passati dai pesci e possono essere osservati visivamente in acqua. Questo ci permette di schermo per il pesce con forte infortunio non letale, che può essere messo in comune per ulteriori sperimentazioni. Minimizzare il numero di pesci illeso o pesce che muoiono prima di raggiungere il punto finale dell&#39;esperimento porta a più uniforme ed efficiente la raccolta di dati e di analisi. Inoltre, non sono richiesti particolari dispositivi o reagenti, rendendo questo metodo conveniente e adatto per l&#39;uso in un ambiente accademico o di insegnamento. Utilizzando il nostro metodo, qui mostriamo effetti sempre maggiori delle dosi gentamicina sulla rigenerazione del rene, nonché l&#39;effetto di aumento della 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.

Infortunio gentamicina può essere confermata visivamente dall&#39;osservazione di calchi epiteliali renali in acqua (Figura 1). Diverse dosi di gentamicina sono stati usati per danneggiare adulto tipo selvatico TuAB zebrafish, con conseguente aumento del numero di lhx1a + aggregati cellulari nel rene rigenerante (Figura 1A - D). Calchi bianche possono essere facilmente visibile in acqua 1 giorno dopo la lesione (Figura 1E - H).</strong...

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.

Rene Rigenerazione in Zebrafish adulti da Gentamicina 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 ...

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

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10.8K Views.  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. 

Video: 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.

Approccio preciso di ablazione cellulare per la modellazione di lesioni acute del rene nello sviluppo di 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 ...

Ricerca

Biologia dello sviluppo

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 Ferita Lesione del Zebrafish adulti Telencefalo: Un metodo per Indagare vertebrati cervello neurogenesi e rigenerazione

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

Neuroscienze

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.

Microiniezioni endovenosa di larve Zebrafish allo Studio danno renale acuto

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.

Modello di lesione da coltellata del tectum ottico adulto utilizzando zebrafish e medaka per l'analisi comparativa della capacità rigenerativa

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.

Il trapianto di cellule direttamente nel rene di Zebrafish adulti

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.

Dissezione del rene Zebrafish adulti

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.

Ablazione laser del Pronephros Zebrafish allo Studio renale rigenerazione epiteliale

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.

Un test per Lateral Rigenerazione Line in Zebrafish adulti

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.

Analisi di Nefrone Composizione e funzione del rene Zebrafish adulti

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.

Modello di lesione renale acuta indotto dal cisplatino nel pesce zebra adulto

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