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 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.

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

Research

JoVE Journal

Developmental Biology

10.7K 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

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.

Mechanical Injury Vessel in embrioni di zebrafish

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

Ricerca

Biologia dello sviluppo

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.

Ablazione specifiche Hepatocyte in Zebrafish allo Studio biliare-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 ...

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.

Sviluppo di un modello di fegato Fibrotic etanolo-indotta in zebrafish per studiare progenitrici cellulo-mediata epatociti Rigenerazione

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 di adulti transgenici Zebrafish retina espianti per live-cell imaging per 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.

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 ...

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.

Analisi istologiche di lesioni al fegato acuto alcolico 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 ...

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.

Un metodo semplificato ed efficiente per isolare i Keratinocytes umani primari dal tessuto pelle adulta

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.

Nanoparticelle-mediata siRNA silenziamento genico nel cuore di 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.

Modelli di lesione di Zebrafish adulto per studiare gli effetti del Prednisolone nella rigenerazione di tessuto osseo

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

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

Metodo di elettroporazione per in vivo Consegna del DNA plasmide nel Telencephalon del pesce zebra adulto

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...