Wir möchten Trent Waugh für seine technische Unterstützung danken. Wir die Abteilung für Pädiatrie an der Klinik erkennen auch für kranke Kinder und Cure kongenitaler Muskeldystrophie (CMD) für die großzügige Finanzierung für dieses Projekt.

1. Herstellung von Agar Injection Plates (Dauer: 45 min) <ol><li> Kochen Sie 2% bis 3% Agarose in E3 Medien und ermöglichen Lösung leicht auf der Bank zu kühlen. Anmerkung: Die Anzahl der Einspritzplatten vorbereitet bestimmt die Menge an Agarose erforderlich. Jedes Injektionsplatte muss ca. 35 ml der Agarose-Lösung. </li><li> Nach dem Auskochen die Agarose zu kühlen, bis die gewünschte Temperatur erreicht ist (z. B. 45 ° C) nach der Spritzgießform Herstellers. </li><li> Gib ca. 35 ml der Agarose in einen 100-mm-Schale abgekühlt. </li><li> Platzieren Sie ein Ende der bevorzugten Spritzgussform in Lösung, dann lag Rest der Form auf Agarose-Lösung (dies wird dazu beitragen, das Auftreten von Luftblasen). </li><li> Damit die Agarose-Lösung entweder bei RT oder bei 4 ° C für ca. 30 min verfestigt. </li><li> Verwenden Sie einen Spachtel, um ein Ende der Form aus dem festen Agarose zu trennen. Entfernen Sie langsam den Rest des m<html> alt. </li></ol> 2. Herstellung von Evans Blue-Farbstoff (EBD) Injection Mix (Dauer: 30 min) <ol><li> Einen 1% Vorrat an EBD in 1X Ringerlösung (155 mM NaCl; 5 mM KCl; 2 mM CaCl 2; 1 mM MgCl 2; 2 mM Na 2 HPO 4, 10 mM HEPES, 10 mM Glucose; pH 7,2), welches bei RT gelagert werden können. </li><li> Machen Sie eine Stammlösung von Fluoresceinisothiocyanat (FITC) -Dextran MW 10.000 kDa bei 25 mg / ml in 1X Ringer-Lösung und bei -20 ° C </li><li> Bereiten Injektionsmischung durch Verdünnen EBD auf 0,1% direkt im Stammlösung von FITC-Dextran Lager (dh für eine endgültige Arbeitsvolumen von 100 &amp; mgr; l. Mischen Sie 10 ul 1% EBD in 90 &amp; mgr; l FITC-Dextran Lager). </li><li> Gründlich Wirbelinjektions Mix (es sollte grün) und vor direktem Licht zu halten durch Umwickeln Injektionsmischrohr mit Aluminiumfolie. </li></ol> 3. EBD Injektionspräparat (Dauer: ca. 30 min) ent &quot;&gt; Hinweis: Protokoll funktioniert am besten mit Larven aus 3-7 Tage nach der Befruchtung (DPF). <ol><li> Pre-warmen Einspritzplatte auf RT. </li><li> Einrichten Einspritzvorrichtung durch Anordnen von Mikromanipulator auf Metallplatte und stehen neben dem Mikroskop wird zur Injektion verwendet. Schalten Sie Luft angetrieben Mikroinjektion Controller. Hinweis: Die bevorzugte Injektionssystem wird von Labor variieren und sollte Ergebnis der Analyse nicht ändern. </li><li> Zurück zu füllen Injektionsnadel mit rund 2-4 ul der EBD-Mix. </li><li> Kalibrieren Injektionsvolumen etwa 5 nl der EBD-Mix. Hinweis: Injektionsvolumen Kalibrierung wird am Kalibrierungsverfahren abhängen. Ein Kolben angetrieben Injektion kann direkt an einen bestimmten Einspritzmenge eingestellt werden, wohingegen Gasdruck-Injektoren die Einspritzmenge über Volumen-Bolus mit dem Einsatz von einem Mikrometer kalibriert benötigen. </li><li> Wet Spritz Platte mit 1X Ringer-Lösung und entfernen Sie überschüssiges aus Brunnen. </li><li> Pre-Treat-Larven, die mit 0,04% Ethyl-3-aminobenzoat Methansulfonat salt (Tricaine) in 1X Ringerlösung Larven vor dem Beginn der Einspritzung zu immobilisieren verdünnt. Hinweis: Die Gewährleistung der Larven sind komplett unbeweglich ist wichtig, da die Injektions ist schwierig mit jeder Restbewegung. </li><li> Zeigen anästhesiert Larven in die Vertiefungen der Agar-Einspritzplatten unter Verwendung einer Glaspipette. Stellen Sie sicher, dass die Larven sind vollständig in den Brunnen und auf ihrer Seite liegen. Anmerkung: Die Zahl der Larven pro Vertiefung bis zu den Experimentator. </li><li> Nach Larven werden in den Vertiefungen setzen, entfernen Sie überschüssige Ringer-Lösung, um Larven Bewegung innerhalb der gut zu minimieren. Lassen Sie eine Restmenge an Lösung, so dass Larven nicht zu dehydrieren. </li></ol> 4. Pericardial Injektion von Zebrafisch-Larven mit EBD (Time: Abhängig von der Anzahl der Larven injizierende, schätzungsweise 1-3 h) <ol><li> Legen Sie die Einspritzplatte, die die Larven auf einem Binokular, wo die Injektionen durchgeführt. </li><li> Positionieren Sie die Injektionspipette Nadel enthält,die EBD-Mix über einen Zebrafisch-Larven. </li><li> Re-Position der Einspritzplatte durch Drehen, so dass die Injektionsnadel in der Nähe Herzen der Larven und etwa 45 ° ventral von der Vorwärts-Rückwärts-Achse. </li><li> Einfügen der Injektionsnadel in die gemeinsame Cardinalvene (CCV) im Bereich der Vene am vorderen Teil des Eigelbs, wo die Ader wird zunächst Drehen in dorsaler Richtung (Figur 1). Hinweis: Eine Vergrößerung von bis zu 40x kann nützlich sein, deutlich sehen, die CCV sein. </li><li> Injizieren Sie 5 nl der EBD-Mix und halten Injektionsnadel in Position für 5-8 Sekunden, um sofortige Leckage von EBD-Mix zu minimieren. Anmerkung: Eine gute Einspritzung Farbstofffärbung in den Herzkammern gesehen (Figur 1) aufweisen. Wenn EBD Mischung nicht im Herz beobachtet, dann Einspritzen einer zusätzlichen 5 nl des EBD Mischung kann ausreichen, um die Farbstoffaufnahme induzieren. Alternativ kann die Embryos verworfen. Hinweis: In einigen Situationen kann der Herzschlag zu stoppen. Wenn diese occurs, weiterhin die Larven für 20-40 sec überwachen. In der Regel nimmt der Herzschlag, wie der Farbstoff durch das Kreislaufsystem bewegt. </li><li> Gehen Sie zum nächsten Larven und wiederholen. </li><li> Identifizierung erfolgreich injizierten Embryonen durch Beobachten der Gegenwart von FITC-Dextran in die Vaskulatur unmittelbar nach der Injektion (Figur 2). </li></ol> 5. Inkubation und EBD Aufnahme (Time: 4-6 h) <ol><li> Nachdem die gewünschte Anzahl von Larven injiziert Rück injiziert Larven auf 1X Ringerlösung ohne Tricaine in 100 mm-Schalen. </li><li> Halten Sie Speisen in Aluminiumfolie eingewickelt. Hinweis: Halten Sie injizierten Larven in der Dunkelheit deutlich erhöht Überlebensraten und gewährleistet die größte Konsistenz in der Signalstärke. Wickel in Aluminiumfolie ist besonders wichtig für die Zeitspanne, die die Larven sich außerhalb des Inkubators. </li><li> Erlauben Larven bei 28,5 ° C für 4-6 Stunden inkubiert, um eine ausreichende EBD Aufnahme zu gewährleisten. </li></ol&gt; 6. Visualisierung der EBD im Muskel (Time: Abhängig von der Anzahl der Larven injizierende und Art der Mikroskopie, geschätzte 0,5 bis 3 h) <ol><li> Vor der Bilderzeugung, betäuben die Larven mit 0,04% Tricaine um Bewegung zu verhindern. </li><li> Ansicht Larven unter rote Fluoreszenz zu bestimmen, ob EBD-Aufnahme in Skelettmuskel (3) auftritt. </li></ol>

<ol>
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Die Autoren haben keine konkurrierenden finanziellen Interessen oder andere Interessenkonflikte.

Zebrafisch als ein leistungsfähiges Werkzeug für die Untersuchung der neuromuskulären Erkrankungen 2,29 Schwellen. Bis heute hat der Zebrafisch-System verwendet worden, um neue Muskel krankheitsverursachende Mutationen 16,17,30 validieren, aufzuklären neue Pathomechanismen 18 und identifizieren potenziell neue Therapeutika 12,24. Diese gemeinsamen Anstrengungen haben den Nutzen der Zebrafisch die menschliche neuromuskuläre Krankheiten Modell etabliert. Doch trotz der Fortschritte mit Zebrafischen und Säugetieren Modellen, gibt es begrenzte Behandlungsmöglichkeiten für Patienten in das breite Spektrum der neuromuskulärer Krankheiten. Daher besteht eine hohe Nachfrage nach Therapieentwicklung für diese Gruppe von verheerenden Krankheiten. Parallel zu dieser Nachfrage nach Therapeutika ist die entsprechende Notwendigkeit einer laufenden experimentellen Innovationen sowie rigorose Analyse, neue Tiermodelle und vermeintlichen therapeutischen Strategien zu überprüfen. EBD-Analyse wird häufig in Maus-Modellen verwendetStudie Gewebe und Zellschäden im Gehirn, Herz und Skelettmuskel 27,31. Am auffälligsten ist, EBD ausgiebig in Mausmodellen von verschiedenen Muskeldystrophie Subtypen verwendet werden, um den Schweregrad der Muskelmembran Instabilität zeigen und 8 beschädigen. Die Verwendung von EBD, um Muskelmembranschäden aufzudecken ist eine unterstützende Parameter Gründung Ähnlichkeiten des Tiermodell für das menschliche Krankheitszustand 9. Die Macht der EBD in Maus hat mehrere Labors, einschließlich unserer eigenen führte, zu entwickeln und anzuwenden, um EBD Zebrafisch Modelle von neuromuskulären Erkrankungen. Aufgrund der Anwendbarkeit der EBD-Analyse wird diese Technik aktiv umgesetzt, um Zebrafisch-Modelle auf den menschlichen Krankheitszustand 11,15,22,24,32 mauern. Larven mit beschädigten Muskelmembranen müssen EBD-Aufnahme und damit rote Fluoreszenz in Muskelfasern. Fluoreszenz in dem Raum zwischen den Fasern beobachtet, jedoch nicht im einzelnen Muskelfasern kann auch informative Faser Ablösen von der Basalmembran in t seiner Abwesenheit von Membranschäden, die nützliche diagnostische Details. EBD-Analyse hat mögliche Anwendung außerhalb Tiermodellvalidierung. Die Bemühungen von unserem Labor haben kürzlich gezeigt, dass EBD-Analyse ist von Vorteil bei der Validierung potenziell neuartigen Therapien 24. Feststellung, ob potenzielle therapeutische Behandlungen reduzieren oder abzuschaffen EBD-Aufnahme in neuromuskuläre Erkrankung Modelle können relevante therapeutische Wirkung 8 bedeuten. Diese Art der Analyse kann helfen, die Mechanismus (n) von Therapeutika und dehnt die Anwendung der EBD-Analyse. Wie bei vielen Techniken, hat EBD Analysen haben mehrere Einschränkungen, die während experimentellen Design und Praxis eingehalten werden. Zum Beispiel kann es schwierig sein, die CCV identifizieren aufgrund der Verdickung des Gewebes mit dem Alter. Zusätzlich ist es einfach zu Larven in Vorbereitung beschädigen vor und während des perikardialen Injektion Reduktions experimentellen Zählungen und erhöhen den Bedarf an einer großen Anzahl von Larven prep.Außerdem können mechanische Schäden die Larven während der Handhabung und Einspritzung erfolgen in false positives Ergebnis als beschädigt Muskel aufnehmen kann EBD. Um einige dieser Hindernisse zu überwinden, haben wir ein Co-Einspritzstrategie in diesem Video-Artikel, der eine einfache und zuverlässige Identifikation von Larven mit sofort erfolgreich Farbstoff Infusion nach der Injektion und vor der anschließenden Analyse erlaubt beschrieben. Die FITC-Dextran Coinjektion Steuerungen für erfolgreiche Injektion indem Bestätigung EBD im Gefäßsystem vor der Aufnahme durch die Muskelfasern. Dies kann besonders nützlich sein, da EBD Fluoreszenz wird nach mehreren Stunden, wenn nicht in den Muskelfasern gesammelt stark diffuse in Larven; Als solches ist es schwierig zu erkennen sein. Darüber hinaus fehlt der CCV und Injizieren EBD in den Dotter oder Körperhöhle kann nach der Inkubation ergeben diffuse rote Fluoreszenz ähnlich Embryonen zu steuern, noch mit der verringerten Wahrscheinlichkeit der Aufnahme durch beschädigte Muskelfasern. Gemeinsam sind diese Höhlenats vorschlagen EBD Injektion erfordert Geduld und Praxis, um konsistente und zuverlässige Ergebnisse zu erzielen. In all beschreiben wir eine praktische und einfache Methode, um EBD-Analyse auf Zebrafisch-Larven durchzuführen. Bisher hat sich die Verwendung von Zebrafisch als Modellsystem, vor allem als Mensch Krankheitsmodell, wurde schnell wachsenden. Diese Expansion ist zum Teil auf die weitere Entwicklung und Veränderung der experimentellen Techniken, die auf den gegenwärtigen Vorteile der Zebrafisch-System zu verbessern. Die EBD Injektionstechnik stellt eine zusätzliche und leistungsfähiges Werkzeug, um eines Forschers Arsenal für die Validierung und Untersuchung der Zebrafisch Muskelkrankheitsmodellen. Die laufende Umsetzung und Änderung dieser Technik hat das Potenzial, zu helfen aufzudecken neuer Therapiestrategien sowie krankheitsverursachenden Mechanismen.

Muscular dystrophies constitute a group of prevalent and heterogeneous human muscle diseases with specific histopathological features1,2. Symptoms typically associated with this devastating group of diseases include muscle weakness, muscle degeneration, loss of motility, and early mortality1,3. The primary pathomechanisms of muscular dystrophies are the loss of proteins that stabilize the sarcolemma, anchor transmembrane complexes, and mediate cell signaling across the membrane4-6. For example, complete loss of the protein dystrophin, a primary scaffold protein of the dystrophin-glycoprotein complex, results in destabilization of the muscle membrane in Duchenne muscular dystrophy7. Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity8,9. This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems2,10-15. However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes. This limitation of viable therapies is due to several key factors: 1) the difficulty of gene therapy strategies, 2) a high frequency of de-novo disease cases and the corresponding lack of translatable animal models, and 3) the lack of rigorous strategies to test the physiological consequences of putative therapeutic agents with clear and reproducible outcome measures.
To overcome some of these limitations, numerous labs including our own are employing zebrafish as a system to model and study human neuromuscular diseases2. To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations16,17, elucidate uncharacterized disease causing mechanisms17,18, and identify novel therapeutic strategies12,19. These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers20. Zebrafish have additionally proven amendable to large-scale drug screens21, a valuable method for the identification of novel therapeutics22-24. Regarding muscle disease research, these strengths are complemented by the ability to isolate single zebrafish skeletal muscle fibers via dissociation25 and by the ability to examine myofiber organization in vivo using the optical phenomenon called birefringence26, which collectively allows for rapid determination of macroscopic muscle integrity. Regardless of these available utilities, further tool development is continuously required to advance investigation.
We, and others, have adapted a protocol for EBD injection and analysis in the zebrafish model. EBD is a vital, cell permeable dye that is taken up by damaged, degenerating, or apoptotic cells and then visualized under fluorescence27. To date, EBD analysis has extensively been used to analyze muscle membrane integrity in mouse models of skeletal muscle and heart diseases8,9,27. However, in mammalian preparations, harvested muscle typically requires laborious sectioning or dissection prior to analysis. In zebrafish, direct analysis is possible in high numbers using live and intact animals. In this video article, we will demonstrate the methodology to perform EBD injection and analysis in live zebrafish larvae, with representative images of EBD uptake in the zebrafish dystrophy mutant line sapje15,28. Furthermore, we present a co-injection strategy that allows for increased quantification of EBD preparations.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fluorescein isothiocyanate-dextran MW 10,000</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">FD10S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Evan&#39;s Blue Dye</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm Petri dish</td>
			<td style="width:173px;">Fischerbrand</td>
			<td style="width:140px;">FB0875712</td>
			<td>Injection mold base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Thin wall glass capillaries</td>
			<td style="width:173px;">World Precision Instruments</td>
			<td style="width:140px;">TW100F-4</td>
			<td>For Injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Agarose</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">AGA001</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microinjection mold</td>
			<td style="width:173px;">Adaptive Science Tools</td>
			<td style="width:140px;">TU-1</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">SOD001</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Potassium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">POC888</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Magnessium chloride hexahydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">M2670</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium phosphate monobasic monohydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">S9638</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">H4034</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glucose</td>
			<td style="width:173px;">BioBasic</td>
			<td style="width:140px;">GB0219</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Calcium chloride</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">C1061</td>
			<td>Ringer&#39;s solution</td>
		</tr>
	</tbody>
</table>

analysis of zebrafish larvae skeletal muscle integrity with evans blue dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Die EBD Injektion Mischung wurde bei 3 dpf in die CCV von sapje homozygote Mutanten und Wildtyp-Geschwister injiziert. Injektionen, die den Herzkammern (1B) gefüllt wurden dann für eine erfolgreiche Injektion durch die Visualisierung von FITC-Dextran in die Vaskulatur unter grüne Fluoreszenz (Figur 2) untersucht. Nach 4 Stunden Inkubationszeit wurde EBD Aufnahme an der Somitenstufe mittels Fluoreszenzmikroskopie untersucht. Wildtypgeschwister zeigte keine EBD Fluoreszenz innerhalb sichtbare Muskelfasern (3A), wohingegen die sapje homozygoten Mutanten zeigten EBD Aufnahme, was Schäden an der Muskelmembran 15 (3B). <img alt="Abbildung 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> Abbildung 1. Spritzen EBD Injektion Mischung in den gemeinsamen Kardinal Vene (CCV) eines Zebrafisch embryo. (A) nicht-injizierten Embryo. Pfeil zeigt den idealen Standort für CCV Injektion. (B) Erfolgreiche Injektion in den CCV. Der Farbstoff dringt in die Herzkammern (Pfeil) und beginnt, durch das Gefäßsystem gepumpt werden. (C) Ein erfolgloser CCV Injektion wird in einigen oder allen der Farbstoff Eingabe der Dottersack des Embryos (Pfeil) zur Folge haben. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53183/53183fig2.jpg" /> Abbildung 2. Die Embryos können für eine erfolgreiche Injektion durch Beobachtung FITC-Dextran Verteilung unter grüne Fluoreszenz im gesamten Gefäßsystem unmittelbar nach der Injektion und vor der EBD Aufnahme sortiert werden.Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. <img alt="Figur 3" src="/files/ftp_upload/53183/53183fig3.jpg" /> Abbildung 3: EBD wird durch Fasern mit beschädigten Membranen (A) Wildtyp Geschwistern zeigt keine EBD Fluoreszenz in Muskelfasern entnommen werden.. (B) Sapje ​​homozygote Mutante mit EBD Fluoreszenz in mehreren Muskelfasern (Pfeile). Alle Larven wurden mit dem EBD Injektions Mischung injiziert und nach 4 h Inkubation bei 3 dpf analysiert. Geschwister und Mutanten wurden durch Muskelfaserablösung vor dem CCV Injektions sortiert. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analyse von Zebrafisch-Larven Skelettmuskel Integrität mit Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

analysis-zebrafish-larvae-skeletal-muscle-integrity-with-evans-blue

Research

JoVE Journal

Developmental Biology

9.0K Aufrufe.  The Hospital for Sick Children. In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Serie: Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Erfassen von Gewebereparatur in Zebrafisch-Larven mit Zeitrafferhellstereomikroskopie

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Forschung

Entwicklungsbiologie

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Die Induktion von Akute Skelettmuskelregeneration durch Cardio Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Mikrostrukturierter Apparate für optimierte Mikroinjektion und Imaging von Zebrafischlarven

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Anwendung der chronische Stimulation kontraktilen Aktivität induzierten Ratte Skelettmuskulatur phänotypische Anpassungen zu studieren

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Kennung des skelettartigen Muskels Satellitenzellen durch Immunfluoreszenz mit Pax7 und Laminin-Antikörper

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

Isolation, Kultur, Charakterisierung und Differenzierung menschlicher Muskelprogenitorzellen aus dem Skelettmuskelbiopsieverfahren

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Durchführung von Human Skeletal Muscle Xenografts bei immundefizienten Mäusen

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolierung und Differenzierung von primären Myoblasten von Mausskelettmuskelexplants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Ganzer Berg Immunhistochemie in Zebrafisch Embryonen und Larven

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

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, ...