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.

Alle tierischen Verfahren wurden von der Ethikkommission der Universität Mie genehmigt und wurden gemäß den japanischen Tierschutzverordnung &quot;Gesetz über die Wohlfahrt und Verwaltung von Animals &#39;(Ministerium für Umwelt, Japan) durchgeführt und mit den internationalen Richtlinien eingehalten werden. 1. Herstellung der Nadel HINWEIS: Alle Experimente wurden unter Vollnarkose durchgeführt, und alle Anstrengungen unternommen wurden, um das Leiden zu minimieren. Um Sterbehilfe, Fische wurden in einem Eis-Wasser-Bad (at ≤4 ° C 5 Teile Eis / 1 Teil Wasser) für ≥20 min eingetaucht. <ol><li> Bereiten Sie die Glas Mikrokapillare Nadeln durch Ziehen einer Glaskapillare 1,0-mm-Außendurchmesser mit einer Nadel puller (Abbildung 1A). </li><li> Schneiden Sie die Spitzen der Nadeln schräg mit einer feinen Schere (1B). 200 &amp; mgr; m (Abbildung 1c) - der ideale Spitzendurchmesser sollte etwa 100 sein. Wenn die Spitze diameter zu schmal ist, wird Blut nicht in die Nadel. </li><li> Aufzulösen Heparin in Kochsalzlösung auf eine Konzentration von 5 mg / ml. </li><li> Legen Sie eine vorgeschnittene Nadel im Objektivrevolver Ende einer Wasserstrahlpumpe Rohranordnung und halten Sie das Mundstück in den Mund, oder schließen Sie die Nadel in einer Glühbirne Spender. Tauchen Sie die Nadelspitze in der Heparin-Lösung und Heparinisierung die Nadel durch Saugen und Blasen durch die Lösung (Abbildung 1D und 1E). Hinweis: Das Ansaugrohr Montage für Zebrafisch Spermien Kryokonservierung 16 verwendet. Lange Gummischlauch kann kein Blut fliegen in den Mund zu stoppen. Setzen eines Filters in der Mitte der Röhre können die Gefahren zu vermeiden. </li><li> Bewahren Sie die heparinisierte Nadeln in einer 10 cm Petrischale und an der Luft trocknen für mindestens 1 Std. Eine große Anzahl von Nadeln im Voraus vorbereitet werden. </li></ol> 2. Anästhesie <ol><li> Bereiten Sie die Anästhesielösung in einem kleinen Kunststoffgehäuse durch Mischen von 200 ml Fischwasser mit 100 ul 2-Phenoxyethanol (2-PE). Endkonzentration der Anästhetika ist 500 ppm. VORSICHT! 2-PE hat eine schnelle Wirkung. </li><li> Entfernen gewünschte Anzahl von Fischen (AB-Stamm) aus dem Kreislauf-System. </li><li> Verwenden Sie ein Netz, um die Fische in die Anästhetika für 1 übertragen - 2 min (1F). Beobachten Sie die Fische nach und nach zu schwimmen, zu verbreiten, die Brustflossen horizontal, Keuchen und haben schnelle Operculum Bewegungen innerhalb 1 min. </li><li> Wie die Zeit vergeht, beobachten die Fische lagen auf dem Boden des Gehäuses und schließlich zu stoppen Schwimmen. Die chirurgische Ebene der Anästhesie wird erreicht, wenn der Fisch stoppt nach Luft zu schnappen und die Operculum Bewegungen sind langsam. An dieser Stelle ist Fisch bereit für die Blutentnahme. </li><li> Mit einem Schaumlöffel heben Sie den narkotisierten Fisch aus dem 2-PE und legen Sie sie vorsichtig auf ein Papiertuch mit den Anästhetika (1G) eingeweicht. Bedecken Sie den Kopf des Fisches mit weichem Tissue-Papier auch mit 2-PE-Lösung getränkt, um Augentrockenheit zu verhindern, und verwenden Sie ein anderes trockenes weiches Seidenpapier vorsichtigtrocknen die Körperoberfläche. </li></ol> 3. Blutentnahme <ol><li> Legen Sie eine heparinisierte Nadel im Objektivrevolver Ende des Ansaugrohr-Baugruppe (oder der Glühbirne Spender) und halten Sie die Mundstückende des Ansaugrohr Montage in den Mund. </li><li> Fassen Sie den Objektivrevolver Ende und die Nadel zusammen, und entfernen Sie vorsichtig die störenden Waagen mit der Nadelspitze. Führen Sie die Nadel in einem 30 - 45 ° Winkel in den Blutsammel Website. Durchstechen des Gastrointestinaltraktes (Figur 1H) vermeiden. Im Falle der Verwendung von Lampenspender, drücken Sie die Lampe mit Daumen und Mittelfinger, und blockieren die Öffnung an der Spitze der Lampe mit dem ersten Finger, und setzen Sie die Nadel wie oben beschrieben. Hinweis: Der Ort für die Blutentnahme ist entlang der Körperachse und hinter dem Anus in dem Bereich des dorsalen Aorta. Der dorsalen Aorta (DA) und die hintere Cardinalvene (PCV) nur ventral zur Wirbelsäule (Abbildung 2). </li><li> Fangen Sie an, die saugenMundstückende des Ansaugrohres Anordnung, wenn die Nadel zu berühren die Wirbelsäule zu spüren. Wenn Blut steigt nicht, bewegen Sie die Nadelspitze auf subtile Weise von Hand, um die Durchblutung zu fördern. Beachten Sie, dass, wenn Blut in die Nadel steigen, sofort aufhören zu zittern und saugen sanft (1I). Im Falle der Verwendung von Lampenspender, den Druck der Glühbirne, um das Blut zu saugen. </li><li> Beachten Sie das Blut langsam pulsatil in die Nadel steigen ohne Absaugung, die wegen der arterielle Blutdruck wahrscheinlich ist. Somit ist es nicht notwendig ist, zu saugen, wenn die Nadel die Arterie richtig eindringt. </li><li> Stoppen Absaugung nach dem geeigneten Volumen Blut gesammelt. Entfernen Sie die Nadel aus dem Fisch und drücken Sie die Einstichstelle mit weichem Tissue-Papier, um Blutungen zu stoppen. Beachten Sie die Blutung zu stoppen nach ca. 10 - 20 sec von Fingerdruck (Abbildung 1J). </li><li> Nachdem die Blutung gestoppt ist, den Fisch sofort wieder zurück zu übertragen, um eine cleein Warmwasser (~ 28 ° C) Tank. Helfen Sie dem Fisch vorsichtig wirbelnden Wasser zu den Kiemen, bis es anfängt zu schwimmen zu erholen. Hinweis: Bewahren Sie bis zu 5 postsampling Fisch in einem 2 l-Tank und verbinden Sie den Tank in den Kreislauf-System, um die Fische mit Sauerstoff zu versorgen. Zugabe von Antibiotika zum Fischwasser nicht erforderlich ist. Pflegen Sie den Fisch mit normaler Haltung und Fütterung. </li><li> Vertreibt das Blut von der Nadel auf eine saubere Fläche von einem Stück Parafilm (Abbildung 1 K). </li><li> Messen Blutzucker mit jeder kommerziellen Hand glucometer (Abbildung 1 l). Anmerkung: Die glucometer verwendet eine Glucose-Dehydrogenase-Flavinadenindinucleotid Elektrode und erfordert ein Probenvolumen von 0,6 &amp; mgr; l. <ol><li> Legen Sie einen Teststreifen vollständig in das Messgerät und direkt berühren Sie den Blutstropfen. Automatisch Beachten Sie die Blutentnahme in den Teststreifen und erhalten die Blutzucker 5 Sekunden auf der Anzeigefläche. Nehmen Sie das Ergebnis und entsorgen Sie den Teststreifen. Benutze einenneuen Teststreifen für jede Messung. </li></ol></li><li> (optionaler Schritt) Nehmen genaue Mengen von Blut durch eine Pipettiervorrichtung und übertragen das Blut in ein Mikrozentrifugenröhrchen für weitere Analysen (Hämoglobin, Triacylglycerin (TG), Gesamtcholesterin, etc.). Wenn nötig, verdünnen das Vollblut von einem Fisch mit Kochsalzlösung. Zentrifugieren der Blutproben für 3 min bei 680 · g bei RT und Ernte der Plasma. Übertragen des Plasmas in ein neues Röhrchen. An dieser Stelle ist es bereit, in biochemischen Analysen verwendet werden. </li></ol>

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The authors have nothing to disclose.

Wir stellen Ihnen hier eine detaillierte Protokoll zur seriellen Blutentnahme von erwachsenen Zebrafisch. Dieses Verfahren ist einfach durchzuführen und wir sie verwenden im Labor auf einer täglichen Basis. Das Blutsammelverfahren zum Einsetzen einer Glaskapillare Nadel in den Zebrafisch der dorsalen Aorta basiert. Während dieses Verfahrens ist es wichtig, vorsichtig zu sein, um nicht zu ablatieren die Wirbelsäule, weil sie das Kriterium für die Suche nach der dorsalen Aorta ist. Die Verringerung der Wirbelsäulenv...

Zebrafische sind gewinnt zunehmend an Popularität als ein wertvolles Modell der menschlichen Krankheiten, weil ihre Organe und Genetik ähnlich denen des Menschen 1,2 sind. Auf dem Gebiet der Entwicklungsbiologie, haben viele Studien gezeigt, dass die Zebrafisch und Menschen zeigen deutliche Ähnlichkeit in der Hämatopoese 3, Blutstillung 4,5 und Myelopoese 6. Erwachsenen Zebrafisch sind ebenfalls für die Untersuchung immunologischer 7, neurodegenerative 8 und Fettleibigkeit bedingte Krankheiten 9, weil dieses Modellorganismus Aktien gemeinsamen Wege mit denen in menschlichen Erkrankungen gestört verwendet. Für Übergewicht und Fettleibigkeit bedingte Krankheiten (Diabetes, Fettleber und nichtalkoholische Steatohepatitis und Atherosklerose), Zebrafisch-Blutzucker und Lipide Ebenen wurden gründlich in mehreren transgenen und ernährungsbedingte Fettleibigkeit Modelle 10-13 untersucht. Wiederholte Entnahme von Blut aus einzelnen Tiere Tiernutzung und Dezember zu reduzierenRease interindividuelle Unterschiede. Jedoch ist wiederholte Probennahme wegen ihrer relativ kleinen Blutvolumen und der Mangel an leicht zugänglichen Gefäßen technisch schwierig bei kleinen Tieren wie Zebrafisch. Verschiedene Methoden für die einmalige Blutentnahme von Zebrafisch entwickelt, obwohl diese Methoden haben ihre eigenen Nachteile, einschließlich Letalität assoziiert Gewebeschäden und begrenzte Blutvolumen. B. 1-5 &amp; mgr; l Blut von einem seitlichen Einschnitt von etwa 0,3 cm in der Länge in dem Bereich des dorsalen Aorta 5 geerntet werden. Enthauptung mit einer Schere durch Schneiden durch den Schultergürtel kann 5-10 &amp; mgr; l Blut 10 zu sammeln. Eine weitere praktische Blutentnahme Methode ist tail Ablation 14. Herzpunktion ist eine mögliche alternative Verfahren für wiederholte Blutentnahme aus dem gleichen Fisch, aber die sehr geringe Menge erhalten (etwa 50 nl) mit diesem Verfahren begrenzt die Anzahl der Analysen, die Leis sein kannMed. 11 Dementsprechend wird ein neues Protokoll benötigt, um wiederholter nicht-tödliche Blutabnahme, die zu kritischer Voraus erforderlich wäre dafür Organismus auf eine Standard-Modellorganismus für menschliche Krankheiten zu ermöglichen. Diese Technik würde zum Testen pharmakologische Reaktion, Entdeckung von Molekular Biomarker für die Diagnose, die Bestimmung der Prognose und die Überwachung von verschiedenen Krankheiten, wie Stoffwechselerkrankungen, degenerativen Erkrankungen und verschiedenen Arten von bösartigen Erkrankungen ermöglicht. Deshalb entwickelte eine minimal-invasive Verfahren zur Gewinnung von Blut von Zebrafischen in Reihe 15. Hier werden wir den Vorgang visuell nachweisen und ein detailliertes Protokoll für diese Technik. Unter Verwendung dieses Verfahrens wurden der normale Wert auf der Grundlage verschiedener Parameter, einschließlich Hämoglobin, Nüchternblutzucker und Lipide im Blut von gesunden erwachsenen Zebrafisch ausgewertet. Zusätzlich haben wir auch bewertet, ob diese Methode eignet sich für Studien, die serielle Proben durch m verlangenBERWACHUNG die zeitlichen Veränderungen der Blutzuckerwerte während der Überfütterung Experimenten.

<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

Wiederholte Blutentnahme ist eine der am häufigsten verwendeten Techniken bei Labortieren durchgeführt. Allerdings hat eine nicht-tödliche Protokoll für die Blutentnahme aus Zebrafisch nicht nachgewiesen. Die bisherigen Verfahren zur Blutentnahme aus Zebrafisch sind tödlich, wie laterale Inzision, Enthauptung und Schwanz Ablation. So haben wir eine neuartige &quot;wiederholte&quot; Blutabnahmeverfahren ein detailliertes Protokoll umreißt dieses Verfahren entwickelt, und heute hier. Dieses Verfahren ist minimal-invasiv und führt zu einer sehr geringen Sterblichkeit (2,3%) für Zebrafisch, wodurch wiederholte Entnahme von Blut aus dem gleichen Individuum. Das maximale Volumen der Blutabnahme ist abhängig vom Körpergewicht der Fische. Das Volumen für wiederholte Entnahme von Blut in Abständen sollte ≤0.4% des Körpergewichts pro Woche oder ≤1% alle 2 Wochen, was durch Messungen der Hämoglobin bewertet wurden sein. Zusätzlich Hämoglobin, Nüchternblutzucker, Plasma Triacylglycerol (TG) und c insgesamtholesterol Ebenen bei männlichen und weiblichen erwachsenen Zebrafisch wurden gemessen. Wir wendeten diese Methode auch, um die Fehlregulation des Glukosestoffwechsels in ernährungsbedingte Fettleibigkeit zu untersuchen. Dieses Blutsammelverfahren wird viele Anwendungen, einschließlich Glucose- und Fettstoffwechsels und hämatologischen Studien, die die Verwendung eines Zebrafischs als menschliche Krankheitsmodell Organismus erhöhen können.

Das Blutsammelverfahren bewirkt minimale Verletzung Zebrafisch (ein &lt;1 mm Punktion; Figur 1J) und ergibt eine sehr niedrige Mortalitätsrate von 2,3%. Wir untersuchten das maximale Volumen an Blut, die von einem einzelnen Fisch gesammelt und ausgewertet werden, die die Beziehung um sein Körpergewicht (Abbildung 3) werden könnte. Wir haben festgestellt, dass der maximale Blutvolumen gesammelt wurden linear mit dem Körpergewicht (R = 0,813) korreliert. Das größte Volumen von Blut ...

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.

Wiederholte Blutentnahme für Bluttests in Adult Zebrafisch

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

35.0K Aufrufe.  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.

Serie: 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.

DNA-Replikation Timing mit Zebrafisch als ein In Vivo Modell-System-Profiling

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

Forschung

Genetik

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 von Spermien zur schnellen Isolierung von Keimbahnveränderungen im Zebrafisch

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-Orbital-Injection in Adult Zebrafisch

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

Biologie

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.

Verwendung der Durchflusszytometrie zum Erkennen und Quantitieren veränderter Blutbildung in den sich entwickelnden Zebrafischen

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

Entwicklungsbiologie

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.

Untersuchung der Muskelregeneration in Zebrafischmodellen von Muskelerkrankungen

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.

Intraperitoneale Injektion in Adult Zebrafisch

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.

Optimierte intravenöse Injektion bei adulten Zebrafischen

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

Krebsforschung

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.

Blutentnahme für Biochemische Untersuchungen an adulten Zebrafisch

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 Adult Zebrafisch

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.

Rückgewinnung von Adult Zebrafisch-Herzen für Hochdurchsatz-Anwendungen

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