Name: repeated blood collection for blood tests in adult zebrafish
Uploaded: 2015-08-30T14:00:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

Tutte le procedure di animali sono stati approvati dal Comitato Etico dell&#39;Università di Mie, e sono state eseguite secondo normativa giapponese benessere degli animali &#39;legge sul Welfare e la gestione degli animali&#39; (Ministero dell&#39;Ambiente del Giappone) e il rispetto delle linee guida internazionali. 1. Preparazione del Needle NOTA: Tutti gli esperimenti sono stati eseguiti in anestesia, e sono stati fatti tutti gli sforzi per ridurre al minimo le sofferenze. Per l&#39;eutanasia, i pesci sono stati immersi in un bagno di acqua e ghiaccio (5 parti ghiaccio / 1 parte di acqua a ≤4 ° C) per ≥20 min. <ol><li> Preparare aghi microcapillari vetro tirando un capillare di vetro 1,0 mm di diametro esterno, con un estrattore ago (Figura 1A). </li><li> Tagliare le punte degli aghi obliquamente con le forbici sottili (Figura 1B). Il diametro della punta ideale dovrebbe essere di circa 100-200 micron (Figura 1C). Se la punta diameter è troppo stretta, il sangue non entrerà l&#39;ago. </li><li> Sciogliere eparina in soluzione salina ad una concentrazione di 5 mg / ml. </li><li> Inserire un ago pretagliato alla fine boccaglio di un gruppo del tubo aspiratore e tenere il boccaglio in bocca, oppure collegare l&#39;ago ad un erogatore lampadina. Immergere la punta dell&#39;ago nella soluzione di eparina, e eparinizzare l&#39;ago per aspirazione e soffia attraverso la soluzione (Figura 1D e 1E). Nota: Il gruppo del tubo di aspirazione è stato utilizzato per zebrafish crioconservazione degli spermatozoi 16. Lungo tubo di gomma in grado di fermare il sangue volare in bocca. Impostazione di un filtro nella metà del tubo può evitare i pericoli. </li><li> Conservare gli aghi eparinizzati in una capsula di Petri di 10 cm e asciugare per almeno 1 ora. Un gran numero di aghi può essere preparata in anticipo. </li></ol> 2. Anestesia <ol><li> Preparare la soluzione di anestetico in un piccolo caso di plastica mescolando 200 ml di acqua pesce con 100 ml di 2-fenossietanolo (2-PE). Concentrazione finale del anestetici è 500 ppm. ATTENZIONE! 2-PE ha un effetto rapido. </li><li> Rimuovere numero desiderato di pesce (ceppo AB) dal sistema di circolazione. </li><li> Utilizzare una rete per trasferire i pesci nelle anestetici per 1-2 min (Figura 1F). Osservare il pesce gradualmente nuotare, diffondere le pinne pettorali orizzontalmente, rantolo, e hanno movimenti rapidi opercolo entro 1 min. </li><li> Come il passare del tempo, osservare i pesci disteso sul fondo della cassa e finalmente smettere nuoto. Il piano di anestesia chirurgica raggiungerà quando il pesce si ferma a boccheggiare e movimenti opercolo sono lenti. A questo punto, il pesce è pronto per la raccolta di sangue. </li><li> Utilizzando uno skimmer sollevare il pesce anestetizzato dal 2-PE e delicatamente appoggiarlo su un tovagliolo di carta imbevuto di anestetici (Figura 1G). Coprire la testa del pesce con la carta dei tessuti molli anche imbevuto con una soluzione 2-PE per prevenire la secchezza degli occhi e utilizzare un&#39;altra carta velina morbido e asciutto per delicatamenteasciugare la superficie del corpo. </li></ol> 3. Il prelievo di sangue <ol><li> Mettere un ago eparinizzata alla fine revolver del gruppo del tubo aspiratore (o il distributore lampadina) e tenere l&#39;estremità portavoce del gruppo del tubo di aspirazione in bocca. </li><li> Afferrare l&#39;estremità revolver e l&#39;ago insieme, e rimuovere con attenzione le squame che interferiscono con la punta dell&#39;ago. Inserire l&#39;ago con un 30 - ° Angolo nel sito di raccolta del sangue 45. Evitare la puntura del tratto gastrointestinale (Figura 1 H). Nel caso di utilizzo di dispenser lampadina, premete la lampadina con pollice e il medio, e di bloccare il foro sulla punta del bulbo con il primo dito, poi inserire l&#39;ago come descritto sopra. Nota: Il sito per la raccolta del sangue è lungo l&#39;asse del corpo e posteriore all&#39;ano nella regione dell&#39;aorta dorsale. L&#39;aorta dorsale (DA) e la vena posteriori cardinale (PCV) sono solo ventrale al dorso (Figura 2). </li><li> Inizia a succhiare ilboccaglio estremità del gruppo del tubo di aspirazione quando l&#39;ago è sentita toccare la colonna vertebrale. Se il sangue non aumenta, spostare la punta dell&#39;ago sottilmente a mano per incoraggiare il flusso sanguigno. Si noti che una volta che il sangue è in aumento l&#39;ago, interrompere immediatamente agitazione e succhiare delicatamente (Figura 1I). In caso di utilizzo di dispenser bulbo, rilasciare la pressione del bulbo per aspirare il sangue. </li><li> Osservare il sangue aumentare lentamente l&#39;ago in modo pulsatile senza aspirazione, che è probabilmente a causa della pressione arteriosa. Pertanto, non è necessario per aspirare se l&#39;ago penetra correttamente l&#39;arteria. </li><li> Smettere di aspirazione dopo il volume appropriato di sangue viene raccolto. Rimuovere l&#39;ago dal pesce e premere il sito di puntura utilizzando carta velina morbido per fermare le emorragie. Osservare la fermata sanguinamento dopo circa 10-20 secondi di pressione delle dita (Figura 1J). </li><li> Dopo l&#39;emorragia viene arrestata, trasferire immediatamente il pesce di nuovo ad un CLEun riscaldamento dell&#39;acqua (~ 28 ° C) del serbatoio. Aiuta il pesce di recuperare rimestando delicatamente acqua verso le branchie fino a quando non comincia a nuotare. Nota: Mantenere fino a 5 postsampling pesci in una vasca 2 L e connessione del serbatoio al sistema di circolazione per ossigenare il pesce. L&#39;aggiunta di antibiotici all&#39;acqua pesce non è necessaria. Mantenere il pesce con custodia normale e l&#39;alimentazione. </li><li> Espellere il sangue dal dell&#39;ago su una superficie pulita di un pezzo di parafilm (Figura 1K). </li><li> Misurare la glicemia utilizzando qualsiasi glucometro portatile commerciale (Figura 1L). Nota: Il glucometro utilizza un glucosio-deidrogenasi flavina adenina dinucleotide elettrodo e richiede un volume del campione di 0,6 ml. <ol><li> Inserire una striscia reattiva completamente nello strumento e toccare direttamente la goccia di sangue. Osservare il prelievo di sangue nella striscia di test automatico e di ottenere il glucosio nel sangue risultato 5 secondi sul display. Registrare il risultato ed eliminare la striscia reattiva. Usa unnuova striscia reattiva per ogni misurazione. </li></ol></li><li> (passaggio facoltativo) Prendere quantità precise di sangue da un pipettaggio e trasferire il sangue in una provetta per ulteriori analisi (emoglobina, trigliceridi (TG), colesterolo totale, ecc.) Se necessario, diluire il sangue intero da un pesce con soluzione salina. Centrifugare i campioni di sangue per 3 min a 680 xg a temperatura ambiente e raccogliere il plasma. Trasferire il plasma in un nuovo tubo. A questo punto è pronto per essere utilizzato nelle analisi biochimiche. </li></ol>

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The authors have nothing to disclose.

Vi presentiamo qui un protocollo dettagliato per l&#39;ottenimento in serie sangue di zebrafish adulto. Questo metodo è semplice da effettuare e lo usiamo in laboratorio su base giornaliera. Questo metodo di raccolta del sangue si basa sull&#39;inserimento di un capillare ago di vetro in aorta dorsale del pesce zebra. Durante questa procedura, è fondamentale stare attenti a non asportare la colonna vertebrale, perché è il criterio per la ricerca per l&#39;aorta dorsale. Ridurre il lesioni alla colonna vertebrale mig...

Zebrafish stanno guadagnando crescente popolarità come un modello valido di malattie umane perché i loro organi e genetica sono simili a quelli degli esseri umani 1,2. Nel campo della biologia dello sviluppo, molti studi hanno dimostrato che il pesce zebra e spettacolo umano contrassegnati somiglianza in ematopoiesi 3, emostasi 4,5, e mielopoiesi 6. Zebrafish adulti sono utilizzati anche per lo studio immunologico 7, 8 e le malattie neurodegenerative legate all&#39;obesità 9 perché questo modello azioni organismo percorsi comuni con quelli perturbato in malattie umane. Per l&#39;obesità e le malattie connesse con l&#39;obesità (diabete, steatosi epatica non alcolica e e aterosclerosi steatoepatite), zebrafish glucosio nel sangue e livelli di lipidi sono state investigate esaurientemente in vari modelli transgenici obesità e dieta-indotta 10-13. Ripetuto prelievo di sangue da singoli animali riduce il consumo animale e dicembrerease differenze interindividuali. Tuttavia, la raccolta dei campioni ripetuto è tecnicamente difficile in piccoli animali come zebrafish causa della loro relativamente piccolo volume di sangue e la mancanza di navi facilmente accessibili. Sono stati sviluppati diversi metodi per un tempo di raccolta del sangue da zebrafish, anche se questi metodi hanno i loro inconvenienti, tra cui letalità, danno tissutale associato e il volume di sangue limitata. Ad esempio, 1-5 microlitri di sangue può essere raccolto da un&#39;incisione laterale di circa 0,3 cm di lunghezza nella regione della aorta dorsale 5. Decapitazione con le forbici tagliando attraverso la cintura pettorale può raccogliere 5-10 microlitri di sangue 10. Un altro comodo metodo di campionamento del sangue è la coda di ablazione 14. Puntura cardiaca è una potenziale metodo alternativo per la raccolta di sangue ripetuto da uno stesso pesce, ma la piccola quantità ottenuta (circa 50 nl) con questa procedura limita il numero di analisi che possono essere effetmed 11. Di conseguenza, è necessario un nuovo protocollo per consentire ripetuto prelievo di sangue non letale, che sarebbe un progresso critica necessaria per questo organismo sia un organismo modello standard per le malattie umane. Questa tecnica consentirebbe di testare risposta farmacologica, la scoperta di biomarcatori molecolari per la diagnosi, la determinazione della prognosi e il monitoraggio di varie malattie, come le malattie metaboliche, malattie degenerative e diversi tipi di tumori maligni. Abbiamo quindi sviluppato un metodo minimamente invasivo per ottenere sangue da zebrafish in serie 15. Qui mostriamo la procedura visivamente e fornire un protocollo dettagliato per questa tecnica. Con questo metodo, il valore normale in base a vari parametri, tra cui l&#39;emoglobina, glicemia a digiuno, e lipidi nel sangue di zebrafish adulti sani sono stati valutati. Inoltre, abbiamo anche valutato se questo metodo è adatto per gli studi che richiedono campioni di serie di mONITORAGGIO i cambiamenti temporali nei livelli di glucosio nel sangue durante gli esperimenti sovralimentazione.

<table><tbody><tr><td>Glass capillaries with filament</td><td>Narishige</td><td>GD-1</td><td>1.0 mm outer diameter.</td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-10</td><td>To produce the needles</td></tr><tr><td>Heparin</td><td>Wako Pure Chemical Industries</td><td>081-00136</td><td>For heparinization</td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td>For blood collection</td></tr><tr><td>Bulb dispenser</td><td>Drummond</td><td>1-000-9000</td><td>For blood collection</td></tr><tr><td>2-phenoxyethanol</td><td>Wako Pure Chemical Industries</td><td>163-12075</td><td>For anesthetizing the fish</td></tr><tr><td>DRI-CHEM3500V</td><td>Fujifilm</td><td>-</td><td>For hemoglobin measurement</td></tr><tr><td>DRI-CHEM Slides</td><td>Fujifilm</td><td>Hb-WII</td><td>For hemoglobin measurement</td></tr><tr><td>Glutest Neo Super</td><td>Sanwa Kagaku Kenkyusho</td><td>-</td><td>For bood glucose measurement</td></tr><tr><td>Wako L-type TG kit</td><td>Wako Pure Chemical Industries</td><td>464-44201</td><td>For TG measurement</td></tr><tr><td>Wako L-type CHO kit</td><td>Wako Pure Chemical Industries</td><td>460-44301</td><td>For total cholesterol measurement</td></tr><tr><td>Parafilm M</td><td>Alcan Packaging</td><td>PM996</td><td>To expel the blood on</td></tr></tbody></table>

repeated blood collection for blood tests in adult zebrafish

Raccolta del sangue ripetuta è una delle tecniche più comuni eseguiti su animali da laboratorio. Tuttavia, non è stato stabilito un protocollo non letale per la raccolta del sangue da zebrafish. I metodi precedenti per la raccolta del sangue da zebrafish sono letali, come incisione laterale, decapitazione e la coda di ablazione. Così abbiamo sviluppato un metodo di raccolta del sangue &quot;ripetuto&quot; romanzo, e qui presenti un protocollo dettagliata che illustra questa procedura. Questo metodo è minimamente invasiva e comporta un tasso di mortalità molto basso (2,3%) per zebrafish, consentendo campionamento ripetuto sangue dello stesso individuo. Il volume massimo di prelievo di sangue dipende dal peso corporeo del pesce. Il volume per ripetuti prelievi di sangue a intervalli dovrebbe essere ≤0.4% del peso corporeo ogni settimana o ≤1% ogni 2 settimane, che sono stati valutati da misurazioni di emoglobina nel sangue. Inoltre, l&#39;emoglobina, glicemia a digiuno, trigliceridi del plasma (TG) e totale eclivelli holesterol in zebrafish adulti maschili e femminili sono stati misurati. Abbiamo applicato anche questo metodo per studiare la disregolazione del metabolismo del glucosio a obesità indotta dalla dieta. Questo metodo di raccolta del sangue consentirà numerose applicazioni, tra cui metabolismo glucidico e lipidico e gli studi ematologiche, che consentirà di aumentare l&#39;uso di zebrafish come un modello di malattia umana organismo.

Questo metodo di raccolta del sangue provoca lesioni minime di zebrafish (un &lt;1 millimetro puntura; Figura 1J) e produce un tasso di mortalità molto basso del 2,3%. Abbiamo esaminato il volume massimo di sangue che potrebbero essere raccolti da un singolo pesce e valutato il rapporto al peso corporeo (Figura 3). Abbiamo trovato che il volume massimo sangue raccolto è linearmente correlata con il peso corporeo (R = 0,813). Il maggior volume di sangue raccolto da un singolo pesce (pe...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Ripetuto il prelievo di sangue per esami del sangue in Zebrafish adulti

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.9K Views.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Video: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Tempo di replicazione del DNA utilizzando Zebrafish come un sistema di modello In Vivo di profilatura

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Ricerca

Genetica

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

Screening dello sperma per il rapido isolamento delle modifiche germinali nel pesce zebra

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-orbitale iniezione in Zebrafish adulti

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Biologia

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Utilizzo della citometria a flusso per rilevare e quantificare la formazione di sangue alterato nel pesce zebra in via di sviluppo

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Biologia dello sviluppo

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Esame della rigenerazione muscolare nei modelli zebrafish di malattia muscolare

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Iniezione intraperitoneale in Zebrafish adulti

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Iniezione endovenosa ottimizzata nel pesce zebra adulto

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Ricerca sul cancro

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Il prelievo di sangue per l&#39;analisi biochimica in Zebrafish Adult

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish adulti

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...