Nous tenons à remercier Bob Nowak (Andor Technology) de l&#39;aide et des conseils techniques. SA-S. est soutenu par une bourse de Heisenberg de la Deutsche Forschungsgemeinschaft (DFG). Ce travail a été soutenu par la DFG subvention SE2016/7-1.

1. Étiquetage cellules isolées Injecter embryons au stade une cellule-avec un plasmide codant pour la GFP ou de toute protéine de fusion GFP-sous le contrôle d&#39;un promoteur β-actine. Au cours du développement, la GFP est alors exprimée d&#39;une façon mosaïque. Ici, nous avons utilisé la construction transgénique Tg [β-actine: α-actinine-GFP], ce qui place la protéine de fusion α-actinine-GFP sous le contrôle du promoteur β-actine. 2. Embedding embryon <ol class="lalpha"><li> Recueillir des embryons de poisson zèbre et de maintenir dans des conditions normales jusqu&#39;au 28 hpf (mise en scène d&#39;après 12). </li><li> Embryons Dechorionate sous le stéréomicroscope avec une pince (Dumont # 5). </li><li> Préparer une lame de microscope avec un anneau de gelée de pétrole. </li><li> Préparer un 1% à bas point de fusion d&#39;agarose / 10% tricaïne solution avec un milieu E3: faire bouillir 100 mg d&#39;agarose dans 10 ml E3 solution pour obtenir un liquide homogène et la solution d&#39;agarose, et aliquote magasinutilisé solution d&#39;agarose à -20 ° C pour une utilisation ultérieure. Pour une utilisation immédiate, laisser la solution bouillie refroidir et conserver au max. 39 ° C. Ajouter la solution mère tricaïne pour obtenir une concentration finale de 10% tricaïne. Les deux tricaïne et d&#39;agarose sont nécessaires pour empêcher des mouvements embryonnaires. </li><li> Intégrer un seul embryon à 1% d&#39;agarose à faible point de fusion / 10% tricaïne / E3 moyenne dans l&#39;anneau de la vaseline, de l&#39;embryon pose naturellement sur le côté, couvrir soigneusement avec une lamelle juste pour maintenir l&#39;embryon en place et d&#39;éviter un convexe surface qui briserait la lumière. Idéalement, le tissu musculaire embryonnaire qui seront ciblés devraient toucher la lamelle. </li></ol> 3. Embryon laser blessures <ol class="lalpha"><li> Préparer le laser micropoint et le microscope: <ol class="lroman"><li> S&#39;il vous plaît se référer au manuel micropoint laser pour obtenir des instructions détaillées sur la façon de configurer et d&#39;utiliser le laser en collaboration avec le microscope. </li><li> Sélectionnez le bonColorant pour la génération d&#39;un faisceau laser de longueur d&#39;onde 435 nm. </li><li> Réglez la puissance du laser en utilisant le curseur d&#39;atténuation. La puissance du laser doit être assez fort pour briser une vitre avec une seule impulsion (mise au point, par exemple sur la lamelle de l&#39;échantillon à tester cette). C&#39;est la puissance du laser nécessaire pour l&#39;ablation de cellules. </li></ol></li><li> Concentrer sur la région d&#39;intérêt ou d&#39;une cellule à l&#39;aide du réticule installé dans l&#39;oculaire du microscope. Utilisez un objectif 20x (40x permettra dommages plus précis, mais souvent la distance de travail ne suffit pas pour cet objectif). </li></ol> Note 1: Le laser ne sera pas seulement de blesser les cellules dans le plan focal, mais aussi des cellules au-dessus et au-dessous qui sont dans le faisceau laser. Assurez-vous avant l&#39;expérience que le laser est parfaitement monté et ajusté pour une mise au point maximale dans l&#39;axe z. Il est préférable de déterminer la profondeur de la blessure au laser avant d&#39;effectuer le test afin de savoir quelle celluleC&#39;est exactement seront blessés. <ol class="lalpha" start="3"><li> Mettre en place la lumière nécessaire à la visualisation de l&#39;expérience: fond clair pour une utilisation normale, ou de fluorescence si le ciblage d&#39;une cellule marquée par fluorescence. </li><li> Envoyer une impulsion de lumière laser à la zone cible, ou de plusieurs impulsions, en fonction du degré souhaité de blessure. </li></ol> 4. Analyse des Traumatismes et de récupération <ol class="lalpha"><li> Si vous le souhaitez, la procédure de blessure laser peut être enregistré en direct (par exemple, en utilisant l&#39;option d&#39;acquisition flux du logiciel Metamorph) 6. </li><li> Immédiatement après la blessure, l&#39;embryon doit être enlevé doucement de l&#39;agarose avec une pince et replacée dans l&#39;eau d&#39;œuf pour la récupération. </li></ol> Note 2: L&#39;activité musculaire (natation et tics) est particulièrement important pour la récupération des muscles squelettiques. Depuis l&#39;embryon ne peut pas bouger à cause de l&#39;exposition tricaïne et l&#39;ancrage rigide agarose, il n&#39;est pas recommended pour maintenir les embryons entre lame et lamelle trop longtemps. <ol class="lalpha" start="3"><li> Après récupération, les embryons peuvent être fixées et analysées comme souhaité par la lumière, la microscopie à fluorescence confocale, ou comme il est indiqué. </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>

Aucun conflit d&#39;intérêt déclaré.

Laser à médiation blessure est une méthode puissante pour infliger des blessures d&#39;une taille désirée par l&#39;ablation des cellules afin d&#39;étudier la régénération dans des conditions contrôlées dans l&#39;embryon de poisson zèbre. Notamment, les cellules peuvent être ciblées avec précision (figure 2) et à la fois le domaine des blessures ainsi que le calendrier peut être commandé. Par la suite, le site de la lésion et les processus de régénération sont facilement contrôl...

<table border="1"><tbody><tr><td> Bloc chauffant </td><td></td><td></td><td></td></tr><tr><td> Paire N ° 5 forceps </td><td> Dumont </td><td></td><td></td></tr><tr><td> Lames de verre </td><td> Menzel </td><td></td><td> 76 x 26 mm </td></tr><tr><td> Lamelles de </td><td> Roth </td><td></td><td> 50 x 24 mm n ° 1 </td></tr><tr><td> Vaseline </td><td></td><td></td><td></td></tr><tr><td> Stéréomicroscope </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> Microscope à fluorescence </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph logiciel </td><td> Dispositifs moléculaires </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Réactifs <ul><li> Faible point de fusion d&#39;agarose (# 50081,Lonza) </li><li> Tricaïne solution mère: 400 mg tricaïne (# A-5040, Sigma-Aldrich) / 100 ml dH 2 O pH 9,0 </li><li> Moyen E3 (5 mM de NaCl, 0,17 mM de 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

Diverses approches expérimentales ont été utilisées dans la souris pour induire une lésion musculaire dans le but d&#39;étudier la régénération des muscles, y compris les injections myotoxine (bupivacaïne, cardiotoxine ou notexin), les transplantations musculaires (dénervation-dévascularisation régénération induite), l&#39;exercice intensif, mais aussi des modèles murins atteints de dystrophie musculaire tels que la souris mdx (pour une revue de ces approches voir 1). Chez le poisson zèbre, les approches génétiques comprennent des mutants qui présentent des phénotypes dystrophie musculaire (tels que runzel 2 ou sapje 3) et antisens morpholinos oligonucléotidiques qui bloquent l&#39;expression des gènes associés à la dystrophie 4. En outre, les approches chimiques sont également possibles, par exemple avec galanthamine, une inhibition de l&#39;acétylcholinestérase composé chimique, ce qui entraîne hypercontraction, ce qui conduit finalement à la dystrophie musculaire 5. Approches Cependant, génétiques et pharmacologiques généralement affecter tous les muscles dans un individu, alors que la gravité des blessures infligées sont physiquement plus facile à contrôler spatialement et temporellement 1. Localisée blessure physique permet l&#39;évaluation de muscle controlatéral comme contrôle interne. En effet, nous avons récemment utilisé laser médiée par ablation cellulaire pour étudier la régénération du muscle squelettique chez l&#39;embryon de poisson zèbre 6, tandis qu&#39;un autre groupe a récemment rapporté l&#39;utilisation d&#39;un laser à deux photons (822 nm) à des dommages très localement la membrane plasmique du muscle du poisson zèbre individu embryonnaire cellules 7. Ici, nous présentons une méthode pour utiliser le laser micropoint (Andor Technology) pour une lésion musculaire squelettique cellulaire dans l&#39;embryon de poisson zèbre. Le laser à micropointes est un laser à haute énergie, qui est adapté pour l&#39;ablation de cellules ciblée à une longueur d&#39;onde de 435 nm. Le laser est relié à un microscope (dans notre configuration, à partir d&#39;un microscope optique Zeiss) de telle sorte que le microscope peut être utilisé à la fois fou focaliser la lumière laser sur l&#39;échantillon et pour visualiser les effets de la blessure (fond clair ou fluorescence). Les paramètres de commande comprennent des impulsions laser de longueur d&#39;onde, de l&#39;intensité et du nombre d&#39;impulsions. En raison de sa transparence et externes du développement embryonnaire, l&#39;embryon du poisson zèbre se prête très bien à la fois induite par laser blessures et pour l&#39;étude de la reprise ultérieure. Entre 1 et 2 jours post-fécondation, les cellules musculaires squelettiques somitiques subir une maturation progressive d&#39;avant en arrière en raison de la progression de somitogenèse du tronc à la queue 8, 9. A ces stades, les embryons se contracter spontanément et de lancer la natation. Le poisson-zèbre a récemment été reconnue comme un organisme modèle vertébré important pour l&#39;étude de la régénération des tissus, comme de nombreux types de tissus (cardiaques, neuronales, vasculaires, etc) peuvent être régénérés après une blessure chez l&#39;adulte poisson zèbre 10, 11.

Blessures laser à médiation a été réalisée sur immobilisées 1 jour embryons âgés. Comme le montre la figure 1, un peu impulsions laser peut générer une petite plaie, facilement reconnaissable par les endommagés, enroulés, actine riches en myofibrilles qui sont normalement tendus entre les limites de somites. Un plus grand nombre d&#39;impulsions laser va cependant conduire à un bloc somite gravement endommagés, où la plupart des myofibrilles sont détruits. Néanmoins, nous pouvons litt?...

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

La méthode présentée ici comprend la blessure précise des embryons de poisson zèbre en direct avec des impulsions laser de haute énergie et l&#39;analyse subséquente de ces blessures et leur récupération avec le temps. Nous montrons aussi comment génétiquement étiquetés simple ou groupes de cellules musculaires squelettiques peuvent être suivis pendant et après la lumière laser dommages induits.

Laser-infligé des blessures de poisson zèbre muscle squelettique embryonnaire

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

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Biology

10.4K vues.  Max Delbrück Center for Molecular Medicine. La méthode présentée ici comprend la blessure précise des embryons de poisson zèbre en direct avec des impulsions laser de haute énergie et l'analyse subséquente de ces blessures et leur récupération avec le temps. Nous montrons aussi comment génétiquement étiquetés simple ou groupes de cellules musculaires squelettiques peuvent être suivis pendant et après la lumière laser dommages induits.

Vidéo: 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.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

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

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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.

Isolement mitochondriale du muscle squelettique

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.

Purification des progéniteurs du muscle squelettique

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 des embryonnaire et larvaire de poisson-zèbre squelettiques fibres musculaires de préparations dissociées

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.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

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.

Électroporation de l’ADN plasmidique dans le muscle squelettique de la souris

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.

Fluoro-respirométrie à haute résolution du muscle squelettique équin

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

Quantification de l’infiltration graisseuse des muscles squelettiques par décellularisation

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.

Pleins feux sur l’auteur : Quantification de l’infiltration graisseuse des muscles squelettiques basée sur la décellularisation  

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.