The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.

Zebrafish sono state sollevate e allevati secondo procedura standard; Esperimenti sono stati approvati dalla University of Pittsburgh Istituzionale Animal Care and Use Committee. 1. Preparazione di embrioni / larve <ol><li> Per condurre accoppiamenti a tempo, impostare adulti maschi e femmine Tg (fabp10a: CFP-NTR) s931 pesci hemizygous o omozigoti O / N e inserire un divisore tra di loro. Rimuovere questo divisore il mattino seguente quando si desidera accoppiamento. Raccogliere gli embrioni da sforzare l&#39;acqua con un colino a maglia fine di plastica. </li><li> Girare il filtro a testa in giù e risciacquare la superficie a rete con un bel flusso di acqua uovo erogata da un flacone. Trasferire 100 embrioni in una capsula di Petri 100 mm con 25 ml di acqua uovo. Mantenere non più di 100 embrioni per capsula di Petri e aumentare a 28 ° C. </li><li> Per inibire la pigmentazione, aggiungere feniltiourea (PTU) nelle capsule di Petri prima di 10 ore dopo la fecondazione (HPF), ed aumentare embryos / larve fino a 80 HPF. La concentrazione finale di PTU è 0,2 mM. ATTENZIONE: PTU è tossico. Utilizzare in conformità con le linee guida appropriate di gestione. </li><li> Anestetizzare le larve aggiungendo 3-amino etil estere benzoico (Tricaine), ad una concentrazione finale di 0,016%, nelle piastre di Petri. Quindi, utilizzando un microscopio a epifluorescenza, larve sorta CFP-positivi. Utilizzare larve con una dimensione del fegato simile e scartare quelli con un fegato più piccolo o più grande. NOTA: qualsiasi larve CFP-positivi, indipendentemente dall&#39;intensità, può essere utilizzato perché non c&#39;è differenza nel grado di epatociti ablazione tra le larve emizigote e omozigote. ATTENZIONE: tricaine è tossico. Utilizzare in conformità con le linee guida appropriate di gestione. </li><li> Rimuovere l&#39;acqua uovo contenente Tricaine e aggiungere acqua uovo integrato con 0,2 PTU mm. Tenere il embrioni / larve a 28 ° C. </li></ol> 2. Mtz Trattamento <ol><li> Preparare fresco soluzione Mtz 10 mM sciogliendo 0,171 g di polvere in 1 MtzAcqua uovo 00 ml integrato con 0,2% PTU mM DMSO e 0,2. Per sciogliere completamente la Mtz, mescolare la soluzione a temperatura ambiente, con una rapida agitazione per 20-30 minuti. Per aiutare solubilizzare Mtz e per migliorare Mtz permeazione in larve, aggiungere DMSO durante la preparazione Mtz. ATTENZIONE: l&#39;esposizione prolungata o un aumento della concentrazione di Mtz è tossico. Utilizzare in conformità con le linee guida appropriate di gestione. </li><li> Separare le larve in gruppi di controllo e sperimentali per il trasferimento di numero desiderato di larve in due diversi 100 mm piatto di Petri o un piatto multi-bene. Per il gruppo sperimentale, mantenere le larve in 10 soluzione Mtz mM; per il gruppo di controllo, tenerli in acqua uovo contenente 0,2% DMSO. </li><li> Coprire le piastre o capsule di Petri contenenti la soluzione Mtz con un foglio di alluminio per evitare che la foto-inattivazione di Mtz, e incubare a 28 ° C. La durata del trattamento Mtz dipende dallo stadio larvale e la linea transgenica. </li><li> Per osservare simile grave ablazione epatociti nella <em&gt; Tg (fabp10a: CFP-NTR) linea, trattare le larve con la soluzione da Mtz da 3,5 giorni dopo la fecondazione (dpf) (per una durata di 36 ore) e da 5 dpf (per una durata di 24 ore). </li></ol> 3. Analisi delle vie biliari-driven rigenerazione del fegato <ol><li> Alla fine del periodo di ablazione, rimuovere la soluzione Mtz dalle piastre. Lavare le larve Mtz trattati aggiungendo 25 ml di acqua uovo. Agitare la piastra 2-3 volte per lavare le larve prima di gettare l&#39;acqua uovo. Ripetete per tre volte e poi immergere le larve di nuovo in acqua uovo. Per evitare edema cardiaca e morte larvale nelle larve Mtz trattati, aggiungere una minore concentrazione di Tricaine, ad una concentrazione finale di 0,008%, per immobilizzare le larve. </li><li> Per l&#39;analisi del fegato, esaminare la dimensione del fegato delle larve Mtz trattati sotto un microscopio a epifluorescenza con il filtro della PCP. Valutare i livelli di ablazione epatociti in base alle dimensioni del fegato relative: grande (non ablazione; 0-10% larve), medio (parzialmente ablated; 10-20%), e molto piccola (completamente ablated; 80-90%). </li><li> Rimuovere le larve con fegati non ablazione o parzialmente ablazione. Tenere larve solo con un minuscolo fegato, che è indicativo di grave ablazione epatociti. Per l&#39;analisi del fegato presso la rigenerazione 0 punto tempo hr (R0h), raccogliere le larve dopo Mtz washout e CFP ordinamento. In caso contrario, mantenere le larve ordinati in acqua uovo integrato con 0,2 PTU mm, fino pronto per la raccolta. </li><li> Trasferire larve raccolte in una provetta da 1,5 ml microcentrifuga e sostituire l&#39;acqua uovo con la soluzione di fissaggio del 3% formaldeide in tampone PEM. Incubare la provetta su un rotatore O / N a 4 ° C. </li><li> Sostituire la soluzione di fissaggio con 1 ml di PBSDT e ruotare il tubo contenente le larve sul rotatore per 5 min. </li><li> Trasferire le larve fissato in una capsula di Petri 100 millimetri contenente PBSDT e usando pinze, rimuovere manualmente tuorlo e pinne pettorali sotto un microscopio da dissezione. Trasferire fino tp 20 larve sezionato in una provetta da 1,5 ml. </li> &lt;li&gt; Sostituisci soluzione PBSDT con 1 ml di soluzione di blocco e Rock il tubo su un rotatore a temperatura ambiente per 2 ore. </li><li> Sostituire soluzione bloccante con 100 ml di soluzione bloccante contenente anticorpi primari. Lentamente rock il tubo su un rotatore O / N a 4 ° C. </li><li> Rimuovere la soluzione di anticorpo primario e lavare con PBSDT 5 volte per 10 minuti ogni volta. Quindi aggiungere 100 ul di soluzione bloccante contenente anticorpi secondari e rock su un rotatore a temperatura ambiente per 2 ore. </li><li> Lavare 5 volte con PBSDT per 10 minuti ogni volta a RT da dondolo su un rotatore. </li><li> Orientare le larve lateralmente su un vetrino e aggiungere una goccia di mezzo di montaggio. Coprirli con cura con un vetro di copertura e sigillare il vetro di copertura con un lucidatore del chiodo. Ora, è pronto per la microscopia confocale. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Grave, ma non mite, epatociti ablazione suscita rigenerazione epatica biliare-driven. Pertanto, dopo il trattamento Mtz e washout, è importante esaminare la dimensione del fegato delle larve Mtz trattato, che può essere valutata intrinseca CFP fluorescenza dal fabp10a: CFP-NTR transgene. Poiché una piccola percentuale di larve Mtz trattati visualizzerà non ablazione (0-5%) o parzialmente ablato (10-20%) fegati, è essenziale per risolvere e solo analizzare le larve con un piccolo fegato, che è indicativo d...

Il fegato è un organo altamente rigenerativa. Durante la rigenerazione del fegato, rigenerante epatociti, le cellule parenchimali del fegato, sono derivati ​​da epatociti preesistenti (rigenerazione del fegato epatociti-driven) o BEC (rigenerazione epatica biliare-driven) 1,2. Danno epatico solito provoca la proliferazione di epatociti preesistenti; tuttavia, quando la proliferazione degli epatociti è compromessa, BEC può contribuire agli epatociti 2-4. Queste due modalità di rigenerazione epatica sono clinicamente significativi. Dopo la rimozione chirurgica di una porzione di fegato umano (ad esempio, a causa di tumori epatici o donatori di fegato vivi), epatociti nel fegato residuo proliferano per recuperare la massa epatica persa. Al contrario, nei pazienti con malattie epatiche gravi, epatociti proliferazione è notevolmente compromessa, in modo che BECs o cellule progenitrici epatiche (LPC) sembrano contribuire a epatociti rigeneranti 5,6. Il modello di epatectomia roditore 2/3 parziale, in cuiepatociti proliferano a recuperare la massa epatica perso, ha contribuito in modo significativo l&#39;attuale comprensione di epatociti-driven rigenerazione epatica 7,8. Tuttavia, non esiste un modello roditore valido che epatociti rigeneranti sono derivati ​​principalmente da BEC. Sebbene diversi modelli di tossina roditori fegato hanno portato all&#39;identificazione di biliare-driven rigenerazione epatica 2-4, recenti studi lineage tracing in topi indicano un contributo minimo di BEC per rigenerare epatociti in questi modelli 9,10. Alcuni dei modelli di pregiudizio roditori fegato, tra cui epatectomia parziale 11-13 e paracetamolo indotto danni al fegato 14,15, sono state applicate a zebrafish e ha portato alla identificazione di nuovi geni o percorsi implicati nella rigenerazione epatica. Tuttavia, epatociti-driven, ma non BEC-driven, la rigenerazione del fegato si verifica in questi modelli danno epatico zebrafish. Pertanto, un modello di danno epatico romanzo in cui BEC contribuiscono ampiamente a regeneratepatociti zione è necessario per una migliore comprensione della rigenerazione epatica BEC-driven. Gli obiettivi generali del modello di ablazione epatociti qui descritte sono (1) per generare un modello di danno epatico nel quale BEC contribuiscono ampiamente a epatociti rigeneranti, e (2) chiarire i meccanismi molecolari e cellulari alla base di rigenerazione epatica BEC-driven. Abbiamo ipotizzato che la gravità del danno determina la modalità di rigenerazione epatica; quindi, abbiamo previsto che la rigenerazione epatica biliare-driven avrebbe avviato su gravi lesioni degli epatociti. Per verificare questa ipotesi, abbiamo sviluppato un modello danno epatico zebrafish generando una linea transgenica, Tg (fabp10a: CFP-NTR) s931, che altamente esprime nitroreduttasi batterica (NTR) fusa con proteina fluorescente ciano (PCP) sotto il fabp10a specifiche epatociti promoter. Poiché NTR converte il profarmaco non tossico, metronidazolo (Mtz), in un farmaco citotossico, che l&#39;ablazione solo destinato NTR-cellule che esprimono 16-18, in questo caso, gli epatociti. Manipolando la durata del trattamento Mtz, il grado di epatociti ablazione può essere controllato. Utilizzando questo modello, abbiamo recentemente riportato che su di una grave perdita epatociti, BEC ampiamente danno luogo a rigeneranti epatociti 19, che è stato ulteriormente confermato da altri due studi indipendenti 20,21. Pertanto, rispetto al suddetto roditori e modelli danno epatico zebrafish, il nostro modello ablazione epatociti è più vantaggiosa per studiare la rigenerazione epatica BEC-driven. Questo protocollo descrive la procedura per eseguire esperimenti di rigenerazione del fegato utilizzando il modello di epatociti di ablazione zebrafish. Questo modello sarà opportuno per determinare i meccanismi alla base di rigenerazione epatica biliare-driven e per schermi chimici per identificare piccole molecole in grado di reprimere o aumentare la rigenerazione del fegato.

<table><tbody><tr><td>3-amino benzoic acid ethyl ester (Tricaine) powder</td><td>Sigma-Aldrich</td><td>A5040</td><td>Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.&amp;nbsp;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Metronidazole</td><td>Sigma-Aldrich</td><td>M1547</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8532</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td></td></tr><tr><td>Calcium sulfate dihydrate</td><td>Sigma-Aldrich</td><td>C3771</td><td></td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>Mounting media (Vectashield)</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>100 x 15 mm petri dish&amp;nbsp;</td><td>Denville Scientific Inc.</td><td>M5300</td><td></td></tr><tr><td>clear nail polish</td><td>Fisher Scientific</td><td>50949071</td><td></td></tr><tr><td>fine forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>Dumont #5&amp;nbsp;</td></tr><tr><td>glass slide</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>glass coverslip</td><td>Fisher Scientific</td><td>12-540-A and 12-542-A</td><td>18 x 18 mm</td></tr><tr><td>zn-5 (mouse anti-Alcam)</td><td>ZIRC</td><td>zn-5</td><td>1:10 dilution</td></tr><tr><td>goat anti-Hnf4a</td><td>Santa Cruz</td><td>sc-6556 (C-19)</td><td>1:50 dilution</td></tr><tr><td>rat anti-RFP</td><td>Allele Biotechnology</td><td>ACT-CM-MRRFP10</td><td>1:300 dilution</td></tr><tr><td>AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)</td><td>Jackson laboratories</td><td>705-605-147</td><td>1:500 dilution</td></tr><tr><td>AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A21240</td><td>1:500 dilution</td></tr><tr><td>Cy3 donkey anti-rat IgG</td><td>Jackson laboratories</td><td>712-165-150</td><td>1:500 dilution</td></tr><tr><td>dissecting microscope</td><td>Leica Microsystems</td><td>S6F</td><td></td></tr><tr><td>epifluorescence microscope</td><td>Leica Microsystems</td><td>M205FA</td><td></td></tr><tr><td>confocal microscope</td><td>Carl Zeiss</td><td>LSM700</td><td></td></tr><tr><td>egg water</td><td></td><td></td><td>0.3 g/L of sea salts &amp;lsquo;Instant Ocean&amp;rsquo; and 0.5 mM CaSO&lt;sub&gt;4 &lt;/sub&gt;in distilled water.&lt;br /&gt;
&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>10X PBS</td><td></td><td></td><td>25.6 g Na2HPO4&amp;middot;7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.&amp;nbsp; Bring to 1 L with distilled water.&amp;nbsp; Adjust pH 7.&amp;nbsp; Autoclave for 20 min at 121&amp;deg;C.</td></tr><tr><td>1X PBSDT</td><td></td><td></td><td>0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.</td></tr><tr><td>PEM buffer</td><td></td><td></td><td>30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.&amp;nbsp; Bring to 1 L with distilled water.&amp;nbsp; Adjust pH 7.</td></tr><tr><td>Blocking solution</td><td></td><td></td><td>Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.</td></tr></tbody></table>

hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration

Il fegato ha una grande capacità di rigenerarsi. Gli epatociti, le cellule parenchimali del fegato, può rigenerare in uno di due modi: rigenerazione epatica hepatocyte- o biliare-driven. In rigenerazione del fegato epatociti-driven, epatociti rigeneranti sono derivati ​​da epatociti preesistenti, mentre, nella rigenerazione biliare-driven, epatociti rigeneranti sono derivati ​​da cellule epiteliali biliari (BECS). Per la rigenerazione del fegato epatociti-driven, ci sono ottimi modelli di roditori che hanno significativamente contribuito alla attuale comprensione della rigenerazione epatica. Tuttavia, non esiste tale modello roditore per la rigenerazione epatica biliare-driven. Recentemente abbiamo riportato su un modello di danno epatico zebrafish in cui BEC ampiamente danno luogo a epatociti sul grave perdita degli epatociti. In questo modello, gli epatociti sono specificamente asportate da un mezzo di farmacogenetica. Qui vi presentiamo in dettaglio i metodi per l&#39;ablazione epatociti e di analizzare il processo di rigenerazione epatica BEC-driven.Questo modello di ablazione specifici epatociti può essere ulteriormente utilizzato per scoprire i meccanismi molecolari e cellulari alla base della rigenerazione epatica biliare-driven. Inoltre, questi metodi possono essere applicati agli schermi chimici per identificare piccole molecole che aumentano o sopprimono rigenerazione epatica.

Trattamento Mtz per 36 ore (A36h, A sta per l&#39;ablazione) 3,5-5 dpf ridotto drasticamente le dimensioni del fegato (Figura 1B). Dopo Mtz washout, considerato come l&#39;inizio della rigenerazione epatica (R), forte fabp10a: CFP-NTR e fabp10a: espressione DsRed riapparso in 30 ore (Figura 1B). La rete biliare intraepatica, valutata mediante espressione di Alcam che è presente nella membrana di BECs 22, inizialmente crollata ma fu ripristinata al 54 hr pos...

Watch this Scientific Journal Video about Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration at JoVE.com

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven 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 ...

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8.5K Views.  University of Pittsburgh. 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.

Video: Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

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.

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 ...

Ricerca

Biologia dello sviluppo

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

L&#39;utilizzo del sistema di Teton per studiare Meccanismi molecolari della rigenerazione Zebrafish

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

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 ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

Ablazione chirurgica Assay per lo studio degli occhi Rigenerazione in Planarie

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

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 ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Analisi del microambiente epatico durante la progressione precoce del carcinoma epatocellulare associato alla steatosi epatica non alcolica nel pesce zebra

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Ricerca sul cancro

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Quantificare le dimensioni del fegato nel pesce zebra Larval utilizzando la microscopia Brightfield

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Profiling dell'espressione genica specifica del tipo di cella nel fegato del mouse

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Biologia

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Epatotectomia parziale nel pesce zebra adulto

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

Targeted Liver Ablation: Inducing Hepatocyte Death with Metronidazole in Transgenic Zebrafish Larvae

Source: Choi T.Y., et al. <a href="https://www.jove.com/v/52785/" target="_blank">Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration</a>. J. Vis. Exp. (2015)
The article describes targeted liver ablation, a method for inducing hepatocyte death with metronidazole in transgenic zebrafish larvae. The sample protocol describes the process for generating, manipulating and assessing targeted liver ablation in zebrafish.

Ablazione epatica mirata: induzione della morte degli epatociti con metronidazolo nelle larve di pesce zebra transgenico

Source: Choi T.Y., et al. Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration. J. Vis. Exp. (2015)
The article describes ...