Wir danken Bob Nowak (Andor Technology) für technische Hilfe und Beratung. SA-S. wird durch ein Heisenberg-Stipendium der Deutschen Forschungsgemeinschaft (DFG) unterstützt. Diese Arbeit wurde von der DFG Gewährung SE2016/7-1 unterstützt.

Ein. Kennzeichnung Einzelzellen Injizieren Eins-Zellen-Embryos mit einem Plasmid oder einem GFP GFP-Fusionsprotein unter der Kontrolle eines β-Actin-Promotor. Während der Entwicklung wird GFP dann in einem Mosaik Weise exprimiert. Hier haben wir das transgene Konstrukt Tg [β-Aktin: α-actinin-GFP], welche Orte die α-Actinin-GFP-Fusionsprotein unter der Kontrolle des β-Aktin-Promotors. 2. Embryo Embedding <ol class="lalpha"><li> Sammle Zebrafischembryonen und pflegen unter normalen Bedingungen bis 28 hpf (Staging nach 12). </li><li> Dechorionate Embryonen unter dem Stereomikroskop mit einer Pinzette (Dumont # 5). </li><li> Bereiten Sie einen Objektträger mit einer Vaseline Ring. </li><li> Eine 1% mit niedrigem Schmelzpunkt Agarose / 10% Tricain Lösung mit E3-Medium: 100 mg Sieden Agarose in 10 ml E3-Lösung, um eine flüssige und homogene Agarose Lösung zu erhalten; Aliquot und lagernunbenutzten Agarose-Lösung bei -20 ° C für die weitere Verwendung. Für den sofortigen Einsatz, lassen Sie die gekochte Lösung abkühlen und halten bei max. 39 ° C. Hinzufügen Tricain Stammlösung, um eine Endkonzentration von 10% Tricain erhalten. Beide Tricain und Agarose sind notwendig, um embryonale Bewegungen zu verhindern. </li><li> Einbetten eines einzigen Embryos in 1% niedrig schmelzender Agarose / 10% Tricain / E3 Medium innerhalb der Vaseline Ring, mit der Embryo über natürlich auf der Seite; vorsichtig mit einem Deckglas abdecken, nur um den Embryo in Position zu halten und um eine konvexe vermeiden Oberfläche, die das Licht brechen würde. Idealerweise sollte der embryonalen Muskelgewebe, die gezielt werden berühren das Deckglas. </li></ol> 3. Embryo Laser-Verwundung <ol class="lalpha"><li> Bereiten Sie die Mikrospitze Laser und Mikroskop: <ol class="lroman"><li> Bitte beachten Sie die Mikrospitze Laser Handbuch für detaillierte Anleitungen zum Einrichten und Verwenden der Laser in Verbindung mit dem Mikroskop. </li><li> Wählen Sie den richtigenFarbstoff zur Erzeugung einer Wellenlänge 435 nm Laserstrahl. </li><li> Passen Sie die Laserleistung durch die Dämpfung Schieberegler. Die Laserleistung sollte stark genug sein, um eine Glasoberfläche mit einem einzigen Impuls (zB Fokus auf dem Deckglas Ihrer Probe, dies zu testen) brechen. Dies ist die Laserleistung für Ablation Zelle erforderlich. </li></ol></li><li> Konzentrieren Sie sich auf die Region oder Zelle von Interesse über das Absehen in das Okular des Mikroskops eingebaut. Verwenden Sie ein 20x-Objektiv (40x wird genauer Schäden ermöglichen, aber oft der Arbeitsabstand ist nicht ausreichend für dieses Objektiv). </li></ol> Anmerkung 1: Der Laser wird nicht nur schädigen Zellen innerhalb der Fokusebene, aber auch Zellen, oberhalb und unterhalb, die innerhalb des Laserstrahls sind. Stellen Sie sicher vor dem Experiment, dass der Laser optimal aufgebaut und angepasst für eine maximale Fokus innerhalb der z-Achse. Am besten ist es, um die Tiefe des Lasers Schädigung vor der Durchführung des Experiments, um zu wissen, zu bestimmen, welche Zelles genau verletzt werden. <ol class="lalpha" start="3"><li> Richten Sie das Licht für die Visualisierung des Experiments benötigt: Hellfeld für den normalen Gebrauch oder Fluoreszenz, wenn Targeting einen fluoreszenzmarkierten Zelle. </li><li> Senden eines Impulses von Laserlicht an die Zielregion oder mehrere Impulse, abhängig von dem gewünschten Grad der Verletzung. </li></ol> 4. Analyse der Verwundung und Recovery <ol class="lalpha"><li> Falls gewünscht, kann das Verfahren der Laser-Schädigung lebenden (zB unter Verwendung des Stroms Akquisition Option des Metamorph Software). 6 aufgezeichnet werden </li><li> Unmittelbar nach der Verletzung sollte der Embryo vorsichtig aus dem Agarose entfernt werden mit einer Pinzette und zurück in Ei Wasser für Erholung gelegt. </li></ol> Anmerkung 2: Muskulös Aktivität (Schwimmen und Zuckungen) ist besonders wichtig für Skelettmuskel Erholung. Da der Embryo kann nicht wegen Tricain Exposition und der steifen Agarose Einbettung zu bewegen, ist es nicht EMPFEHLUnded die Embryonen zwischen Objektträger und Deckglas zu lange zu halten. <ol class="lalpha" start="3"><li> Nach der Bergung kann der Embryo fixiert und analysiert werden, wie gewünscht durch Licht-, Fluoreszenz-oder konfokalen Mikroskopie, wie angegeben. </li></ol>

<ol>
	<li>Charge, S. B., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+regulation+of+muscle+regeneration.">Cellular and molecular regulation of muscle regeneration.</a> Physiol. Rev. 84, 209-238 (2004).</li><li>Steffen, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+runzel+muscular+dystrophy+is+linked+to+the+titin+gene.">The zebrafish runzel muscular dystrophy is linked to the titin gene.</a> Dev. Biol. 309, 180-192 (2007).</li><li>Bassett, D., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+zebrafish+model+of+muscular+dystrophy.">Identification of a zebrafish model of muscular dystrophy.</a> Clin. Exp. Pharmacol. Physiol. 31, 537-540 (2004).</li><li>Kawahara, G., Serafini, P. R., Myers, J. A., Alexander, M. S., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+dysferlin+by+morpholino+knockdown.">Characterization of zebrafish dysferlin by morpholino knockdown.</a> Biochem. Biophys. Res. Commun. 413, 358-363 (2011).</li><li>Behra, M., Etard, C., Cousin, X., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+mutants+to+identify+secondary+target+effects+of+acetylcholine+esterase+inhibitors.">The use of zebrafish mutants to identify secondary target effects of acetylcholine esterase inhibitors.</a> Toxicol. Sci. 77, 325-333 (2004).</li><li>Otten, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xirp+proteins+mark+injured+skeletal+muscle+in+zebrafish.">Xirp proteins mark injured skeletal muscle in zebrafish.</a> PLoS One. 7, e31041 (2012).</li><li>Roostalu, U., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+molecular+interactions+at+damaged+sarcolemma.">In vivo imaging of molecular interactions at damaged sarcolemma.</a> Dev. Cell. 22, 515-529 (2012).</li><li>Stickney, H. L., Barresi, M. J., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somite+development+in+zebrafish.">Somite development in zebrafish.</a> Dev. Dyn. 219, 287-303 (2000).</li><li>Stellabotte, F., Dobbs-McAuliffe, B., Fernandez, D. A., Feng, X., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+somite+cell+rearrangements+lead+to+distinct+waves+of+myotome+growth.">Dynamic somite cell rearrangements lead to distinct waves of myotome growth.</a> Development. 134, 1253-1257 (2007).</li><li>Choi, W. Y., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration.">Cardiac regeneration.</a> Curr. Top. Dev. Biol. 100, 319-344 (2012).</li><li>Stewart, S., Stankunas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+dedifferentiation+provides+replacement+tissue+during+zebrafish+fin+regeneration.">Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration.</a> Dev. Biol. 365, 339-349 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. , 203-253 (1995).</li></ol>

Keine Interessenskonflikte erklärt.

Laser-vermittelte Verletzung ist eine leistungsfähige Methode zur zuzufügen Wunden einer gewünschten Größe durch Abtragen Zellen, um die Regeneration unter kontrollierten Bedingungen in der Zebrafisch-Embryos zu studieren. Bemerkenswert ist, können die Zellen genau ausgerichtet werden (Abbildung 2) und beide die Verletzung Bereich sowie Zeitpunkt gesteuert werden kann. Anschließend werden die verletzte Stelle und Regeneration Prozesse einfach überwacht, aufgezeichnet (Abbildung 3)</stron...

<table border="1"><tbody><tr><td> Heizblock </td><td></td><td></td><td></td></tr><tr><td> Paar Nr. 5 Zangen </td><td> Dumont </td><td></td><td></td></tr><tr><td> Glasobjektträger </td><td> Menzel </td><td></td><td> 76 x 26 mm </td></tr><tr><td> Deckgläser </td><td> Roth </td><td></td><td> 50 x 24 mm # 1 </td></tr><tr><td> Vaselin </td><td></td><td></td><td></td></tr><tr><td> Stereomikroskop </td><td> Leica </td><td></td><td> MZFLIII </td></tr><tr><td> Micropoint Laser </td><td> Andor Technology </td><td></td><td></td></tr><tr><td> Fluoreszenzmikroskop </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph-Software </td><td> Molecular Devices </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Reagenzien <ul><li> Niedrigschmelzende Agarose (# 50081,Lonza) </li><li> Tricaine Stammlösung: 400 mg Tricaine (# A-5040, Sigma-Aldrich) / 100 ml dH 2 O, pH 9,0 </li><li> E3 Medium (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl 2, 0,33 mM MgSO 4) </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

Verschiedenen experimentellen Ansätze wurden in Maus verwendet worden, um Muskelverletzung mit dem Ziel, Muskelregeneration studieren, einschließlich myotoxin Injektionen (Bupivacain, Cardiotoxin oder notexin), Muskel Transplantationen (Denervierung-devascularization induzierte Regeneration), intensive Bewegung zu induzieren, sondern auch murine Modelle Muskeldystrophie wie der mdx-Maus (für eine Überprüfung dieser Ansätze siehe 1). Im Zebrafisch beinhalten genetische Ansätze Mutanten, die Phänotypen Muskeldystrophie (wie Runzel 2 oder sapje 3) aufweisen und Antisense-Oligonukleotid, das die Expression Morpholinos der Dystrophie-assoziierten Genen 4 blockieren. Außerdem sind chemische Ansätze auch möglich, z. B. mit Galanthamin, eine chemische Verbindung Hemmen Acetylcholinesterase, wodurch hypercontraction, die schließlich führt zu Muskeldystrophie 5. Allerdings, genetische und pharmakologische Ansätze generally betreffen alle Muskeln innerhalb eines einzelnen, während das Ausmaß der körperlich zugefügten Verletzungen leichter kontrolliert werden räumlich und zeitlich ein. Lokalisierte Körperverletzung erlaubt die Beurteilung der kontralateralen Muskel als interne Kontrolle. Tatsächlich haben wir kürzlich Laser-vermittelte Zell-Ablation zur Skelettmuskel Regeneration im Zebrafischembryo 6 studieren, während eine andere Gruppe kürzlich berichteten über die Verwendung eines Zwei-Photonen-Laser (822 nm) bis sehr lokal beschädigt Plasmamembran der einzelnen embryonalen Zebrafisch Muskel Zellen 7. Hier berichten wir über eine Methode für die Verwendung der Mikrospitze Laser (Andor Technology) für Skelettmuskel Zellschädigung im Zebrafisch. Die Mikrospitze Laser ein Hochenergielaser die geeignet Zielzelle Ablation bei einer Wellenlänge von 435 nm ist. Der Laser ist mit einem Mikroskop verbunden (in unserem Setup, einem optischen Mikroskop von Zeiss) auf solche Weise, dass das Mikroskop gleichzeitig f genutzt werden kannoder Fokussierung des Laserlichts auf der Probe und zur Visualisierung der Auswirkungen der Verwundung (Hellfeld oder Fluoreszenz). Die Parameter zur Steuerung Laserpulse umfassen Wellenlänge, Intensität und Anzahl der Impulse. Aufgrund seiner Transparenz und externe Embryonalentwicklung, ist die Zebrafisch-Embryos sehr zugänglich sowohl für Laser-induzierten Verletzungen und zur Untersuchung der anschließenden Erholung. Zwischen 1 und 2 Tagen nach der Befruchtung, somitic Skelettmuskelzellen fortschreitend unterziehen Reifung von anterior nach posterior durch die Progression der Somitogenese aus dem Kofferraum zum Heck 8, 9. Bei diesen Stufen, Embryonen spontan zucken und zu initiieren Schwimmen. Der Zebrafisch vor kurzem als ein wichtiges Wirbeltier Modellorganismus zur Untersuchung der Geweberegeneration erkannt worden, da viele Arten von Geweben (Herz, neuronale, vaskulären etc.) kann nach einer Verletzung im erwachsenen Zebrafisch 10, 11 regeneriert werden.

Laser-vermittelte Verletzung wurde an immobilisiertem 1 Tag alten Embryonen durchgeführt. Wie in 1 gezeigt, kann ein paar Laserimpulse erzeugen eine kleine Wunde, leicht erkennbar an der beschädigten, gedreht, Actin-reiche Myofibrillen, die normalerweise zwischen den somite Grenzen gestreckt wird. Eine höhere Anzahl von Laserpulsen wird jedoch in einem massiv geschädigt somite Block, wo die meisten Myofibrillen zerstört führen. Dennoch können wir buchstäblich beobachten, dass &quot;Zeit alle Wun...

Watch this Scientific Journal Video about Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle at JoVE.com

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Die hier vorgestellte Methode umfasst die genaue Verletzung des Lebens Zebrafischembryonen mit hochenergetischer Laserpulse und der anschließenden Analyse dieser Verletzungen und deren Wiederherstellung mit der Zeit. Wir zeigen auch, wie genetisch markierten einzelne oder Gruppen von Skelettmuskelzellen kann während und nach der Laser-Licht induzierten Schäden verfolgt werden.

Laser-Schädigung der Zebrafisch Embryonale Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

laser-inflicted-injury-of-zebrafish-embryonic-skeletal-muscle

Research

JoVE Journal

Biology

10.4K Aufrufe.  Max Delbrück Center for Molecular Medicine. Die hier vorgestellte Methode umfasst die genaue Verletzung des Lebens Zebrafischembryonen mit hochenergetischer Laserpulse und der anschließenden Analyse dieser Verletzungen und deren Wiederherstellung mit der Zeit. Wir zeigen auch, wie genetisch markierten einzelne oder Gruppen von Skelettmuskelzellen kann während und nach der Laser-Licht induzierten Schäden verfolgt werden.

Serie: Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulten und embryonalen Skelettmuskel Microexplant Kultur und Isolation der Skelettmuskulatur Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Forschung

Biologie

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondriale Isolation von der Skelettmuskulatur

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Reinigung von Vorläufern aus Skelettmuskeln

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

Analyse der embryonalen Zebrafisch-Larven-und Skelett Muskelfasern aus Dissoziierte Vorbereitungen

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

Elektroporation von Plasmid-DNA in die Skelettmuskulatur der Maus

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Hochauflösende Fluoro-Respirometrie der Skelettmuskulatur von Pferden

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

Quantifizierung der Fettinfiltration der Skelettmuskulatur durch Dezellularisierung

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Autor im Rampenlicht: Dezellularisierungsbasierte Quantifizierung der Fettinfiltration der Skelettmuskulatur  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.