Nous tenons à remercier Trent Waugh pour son aide technique. Nous reconnaissons également le département de pédiatrie de l&#39;Hôpital pour enfants malades et Cure dystrophie musculaire congénitale (CMD) pour leur généreux financement de ce projet.

1. Préparation de plaques de gélose d&#39;injection (Durée: 45 min) <ol><li> Faire bouillir 2% à 3% d&#39;agarose dans les médias E3 et laisser la solution refroidir légèrement sur le banc. Note: Le nombre de plaques d&#39;injection en cours de préparation dicte la quantité d&#39;agarose requis. Chaque plaque d&#39;injection a besoin d&#39;environ 35 ml de la solution d&#39;agarose. </li><li> Après l&#39;ébullition, laisser refroidir l&#39;agarose jusqu&#39;à ce que la température désirée est atteinte (par ex., 45 ° C) selon les instructions du fabricant de moule d&#39;injection. </li><li> Verser environ 35 ml de la refroidies agarose dans un plat de 100 mm. </li><li> Placez une extrémité du moule d&#39;injection préférée dans la solution, puis posez reste de moule sur une solution d&#39;agarose (cela va aider à réduire l&#39;apparition de bulles d&#39;air). </li><li> Laisser la solution d&#39;agarose se solidifie soit à la température ambiante ou 4 ° C pendant environ 30 min. </li><li> Utiliser une spatule pour séparer une extrémité du moule à partir de l&#39;agarose solide. Retirez lentement le reste de la m<html> Agé de. </li></ol> 2. Préparation du colorant bleu Evans (EBD) Injection Mix (Durée: 30 min) <ol><li> Faire un 1% stock d&#39;EBD dans une solution de 1X Ringer (NaCl mM 155; KCl 5 mM, CaCl2 2 mM; 1 mM MgCl 2; 2 mM Na 2 HPO 4; HEPES 10 mM, 10 mM de glucose; pH à 7,2), qui peuvent être stockées à température ambiante. </li><li> Faire une solution de réserve de l&#39;isothiocyanate de fluorescéine (FITC) -dextrane MW 10.000 kDa à 25 mg / ml dans la solution et magasin de 1X Ringer à -20 ° C </li><li> Préparer mélange d&#39;injection en diluant EBD à 0,1% directement dans la solution mère de stock FITC-dextran (ie, pour un volume de travail final de 100 pi. Mélanger 10 pi de 1% EBD dans 90 ul d&#39;actions dextran FITC). </li><li> Bien agiter au vortex mélange d&#39;injection (il doit devenir vert) et de garder hors de la lumière directe en enroulant tube de mélange d&#39;injection dans une feuille d&#39;aluminium. </li></ol> 3. Injection Préparation EBD (Durée: Environ 30 min) ent &quot;&gt; Remarque: Protocole fonctionne mieux avec des larves de 3-7 jours après la fécondation (DPF). <ol><li> Préchauffer plaque d&#39;injection à la température ambiante. </li><li> Mettre en place des appareils d&#39;injection en organisant micromanipulateur sur la plaque de métal et se tenir à côté du microscope étant utilisé pour l&#39;injection. Tournez sur le contrôleur de micro-injection d&#39;air entraîné. Remarque: Le système d&#39;injection préférée varie en laboratoire et ne devrait pas changer résultats de l&#39;analyse. </li><li> Retour combler aiguille d&#39;injection à environ 2-4 pi de EBD mix. </li><li> Calibrer volume d&#39;injection à environ 5 nl de EBD mix. Remarque: le calibrage de volume d&#39;injection dépendra de la méthode d&#39;étalonnage. Une injection à pistons entraînés peut être directement fixé à un volume d&#39;injection étant donné que les injecteurs à pression de gaz devront le volume d&#39;injection calibrée par le volume de bolus à l&#39;aide d&#39;un micromètre. </li><li> Plaque d&#39;injection humide avec une solution de Ringer 1X et enlever l&#39;excès de puits. </li><li> Larves pré-traitement avec 0,04% éthyl-3 benzoate méthanesulfonate salt (tricaïne) dilué dans une solution de Ringer pour immobiliser 1X larves avant le début de l&#39;injection. Remarque: Assurer larves sont complètement immobiles est important que l&#39;injection correcte est difficile avec un mouvement résiduel. </li><li> Placer les larves anesthésié dans des puits des plaques de gélose d&#39;injection à l&#39;aide d&#39;une pipette en verre. Assurez-vous que les larves sont complètement à l&#39;intérieur du puits et couché sur le côté. Remarque: Le nombre de larves par puits est à l&#39;expérimentateur. </li><li> Après larves sont mis dans les puits, enlever la solution de Ringer excès pour minimiser le mouvement de larves dans le puits. Laissez une quantité résiduelle de solution pour que les larves ne déshydrate pas. </li></ol> 4. injection péricardique de poisson zèbre larves avec EBD (Durée: en fonction du nombre de larves injection, environ 1-3 h) <ol><li> Placer la plaque d&#39;injection contenant les larves sur un microscope à dissection où les injections seront réalisées. </li><li> Placez l&#39;aiguille de pipette d&#39;injection contenantl&#39;EBD mélanger sur une larves de poisson zèbre. </li><li> Repositionner la plaque d&#39;injection en le faisant tourner de sorte que l&#39;aiguille d&#39;injection se trouve à proximité du coeur du larves et environ 45 ° ventralement à partir de l&#39;axe antéro-postérieur. </li><li> Insérez l&#39;aiguille d&#39;injection dans la veine cardinale commune (CCV) dans la région de la veine à la partie antérieure du jaune où la veine est initialement tourne dans le sens de la dorsale (Figure 1). Remarque: Un grossissement de 40x jusqu&#39;à peut être utile de voir clairement la CCV. </li><li> Injecter 5 nl de EBD mélange et de garder aiguille d&#39;injection en position de 5-8 sec pour minimiser les fuites immédiate de EBD mix. Remarque: Une bonne injection aura coloration colorant vu dans les cavités cardiaques (figure 1). Si EBD mélange est pas observé dans le cœur, puis l&#39;injection d&#39;un 5 nl de EBD mélange supplémentaire peut être suffisante pour induire l&#39;absorption de colorant. En variante, l&#39;embryon peut être mis au rebut. Remarque: Dans certaines situations, le cœur peut s&#39;arrêter de battre. Si ce occurs, continuera à surveiller les larves pour les 20-40 sec. Typiquement, le cœur battant reprend le colorant se déplace à travers le système circulatoire. </li><li> Passer à la prochaine larves et répétez. </li><li> Identifier les embryons injectés avec succès par l&#39;observation de la présence de FITC-dextran dans le système vasculaire immédiatement après l&#39;injection (figure 2). </li></ol> 5. Incubation et EBD absorption (Durée: 4-6 h) <ol><li> Après le nombre désiré de larves sont injectés, retour injecté larves à la solution de Ringer 1X sans tricaïne en boîtes de 100 mm. </li><li> Gardez plats enveloppés dans une feuille d&#39;aluminium. Remarque: Garder larves injecté dans l&#39;obscurité augmente considérablement les taux de survie et assure la plus grande cohérence dans la force du signal. Emballage en feuille d&#39;aluminium est particulièrement important pour la période de temps que les larves sont à l&#39;extérieur de l&#39;incubateur. </li><li> Laisser larves à incuber à 28,5 ° C pendant 4-6 heures pour assurer l&#39;absorption d&#39;EBD suffisante. </li></ol&gt; 6. Visualisation de EBD dans le muscle (Durée: en fonction du nombre de larves injection et le type de microscopie, estimé 0,5-3 h) <ol><li> Avant imagerie, anesthésier les larves avec 0,04% tricaïne pour empêcher le mouvement. </li><li> Voir larves en fluorescence rouge pour déterminer si EBD absorption se produit dans le muscle squelettique (Figure 3). </li></ol>

<ol>
	<li>Verma, S., Anziska, Y., Cracco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Duchenne+muscular+dystrophy+(DMD)+for+the+pediatricians+in+the+community.">Review of Duchenne muscular dystrophy (DMD) for the pediatricians in the community.</a> Clin Pediatr (Phila. 49, 1011-1017 (2010).</li><li>Gibbs, E. M., Horstick, E. J., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+into+prominence:+the+zebrafish+as+a+valuable+tool+for+studying+human+myopathies+and+muscular+dystrophies).">Swimming into prominence: the zebrafish as a valuable tool for studying human myopathies and muscular dystrophies).</a> FEBS J. 280, 4187-4197 (2013).</li><li>Lancet, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , 381-845 (2013).</li><li>Lapidos, K. A., Kakkar, R., McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dystrophin+glycoprotein+complex:+signaling+strength+and+integrity+for+the+sarcolemma.">The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma.</a> Circ Res. 94, 1023-1031 (2004).</li><li>Campbell, K. P., Kahl, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+dystrophin+and+an+integral+membrane+glycoprotein.">Association of dystrophin and an integral membrane glycoprotein.</a> Nature. 338, 259-262 (1989).</li><li>Cohn, R. D., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+muscular+dystrophies.">Molecular basis of muscular dystrophies.</a> Muscle Nerve. 23, 1456-1471 (2000).</li><li>Yoshida, M., Ozawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+complex+anchoring+dystrophin+to+sarcolemma.">Glycoprotein complex anchoring dystrophin to sarcolemma.</a> J Biochem. 108, 748-752 (1990).</li><li>Straub, V., Rafael, J. A., Chamberlain, J. S., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+muscular+dystrophy+show+different+patterns+of+sarcolemmal+disruption.">Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.</a> J Cell Biol. 139, 375-385 (1997).</li><li>Matsuda, R., Nishikawa, A., Tanaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+dystrophic+muscle+fibers+in+mdx+mouse+by+vital+staining+with+Evans+blue:+evidence+of+apoptosis+in+dystrophin-deficient+muscle.">Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle.</a> J Biochem. 118, 959-964 (1995).</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>Gupta, V., 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=The+zebrafish+dag1+mutant:+a+novel+genetic+model+for+dystroglycanopathies.">The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies.</a> Hum Mol Genet. 20, 1712-1725 (2011).</li><li>Kawahara, G., 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=Drug+screening+in+a+zebrafish+model+of+Duchenne+muscular+dystrophy.">Drug screening in a zebrafish model of Duchenne muscular dystrophy.</a> Proc Natl Acad Sci U S A. 108, 5331-5336 (2011).</li><li>Guyon, J. R., 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=Modeling+human+muscle+disease+in+zebrafish.">Modeling human muscle disease in zebrafish.</a> Biochim Biophys Acta. 1772, 205-215 (2007).</li><li>Cavanna, J. S., 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=Molecular+and+genetic+mapping+of+the+mouse+mdx+locus.">Molecular and genetic mapping of the mouse mdx locus.</a> Genomics. 3, 337-341 (1988).</li><li>Bassett, D. I., 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=Dystrophin+is+required+for+the+formation+of+stable+muscle+attachments+in+the+zebrafish+embryo.">Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo.</a> Development. 130, 5851-5860 (2003).</li><li>Horstick, E. J., 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=Stac3+is+a+component+of+the+excitation-contraction+coupling+machinery+and+mutated+in+Native+American+myopathy.">Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy.</a> Nat Commun. 4, (1952).</li><li>Davidson, A. E., 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=Novel+deletion+of+lysine+7+expands+the+clinical,+histopathological+and+genetic+spectrum+of+TPM2-related+myopathies.">Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.</a> Brain. 136, 508-521 (2013).</li><li>Telfer, W. R., Nelson, D. D., Waugh, T., Brooks, S. V., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neb:+a+zebrafish+model+of+nemaline+myopathy+due+to+nebulin+mutation.">Neb: a zebrafish model of nemaline myopathy due to nebulin mutation.</a> Dis Model Mech. 5, 389-396 (2012).</li><li>Dowling, J. J., 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=Oxidative+stress+and+successful+antioxidant+treatment+in+models+of+RYR1-related+myopathy.">Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy.</a> Brain. 135, 1115-1127 (2012).</li><li>Detrich, H. W., Westerfield 3rd, ., M, L. I., Zon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Overview of the Zebrafish system. Methods Cell Biol. 59, 3-10 (1999).</li><li>Whitfield, T. T., 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=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral.">Mutations affecting development of the zebrafish inner ear and lateral.</a> , 123-241 (1996).</li><li>Kawahara, G., Guyon, J. R., Nakamura, Y., 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=Zebrafish+models+for+human+FKRP+muscular+dystrophies.">Zebrafish models for human FKRP muscular dystrophies.</a> Hum Mol Genet. 19, 623-633 (2010).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nat Commun. 4, (2013).</li><li>Waugh, T. A., 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=Fluoxetine+prevents+dystrophic+changes+in+a+zebrafish+model+of+Duchenne+muscular+dystrophy.">Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy.</a> Hum Mol Genet. 23 (17), 4651-4662 .</li><li>Horstick, E. J., Gibbs, E. M., Li, X., Davidson, A. E., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+embryonic+and+larval+zebrafish+skeletal+myofibers+from+dissociated+preparations.">Analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations.</a> J Vis Exp. 50259, (2013).</li><li>Smith, L. L., Beggs, A. H., Gupta, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+skeletal+muscle+defects+in+larval+zebrafish+by+birefringence+and+touch-evoke+escape+response+assays.">Analysis of skeletal muscle defects in larval zebrafish by birefringence and touch-evoke escape response assays.</a> J Vis Exp. 50925, (2013).</li><li>Hamer, P. W., McGeachie, J. M., Davies, M. J., Grounds, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evans+Blue+Dye+as+an+in+vivo.+marker+of+myofibre+damage:+optimising+parameters+for+detecting+initial+myofibre+membrane+permeability.">Evans Blue Dye as an in vivo. marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.</a> J Anat. 200, 69-79 (2002).</li><li>Guyon, J. R., 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=Genetic+isolation+and+characterization+of+a+splicing+mutant+of+zebrafish+dystrophin.">Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin.</a> Hum Mol Genet. 18, 202-211 (2009).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> J Clin Invest. 122, 2337-2343 (2012).</li><li>Majczenko, K., 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=Dominant+Mutation+of+CCDC78+in+a+Unique+Congenital+Myopathy+with+Prominent+Internal+Nuclei+and+Atypical+Cores.">Dominant Mutation of CCDC78 in a Unique Congenital Myopathy with Prominent Internal Nuclei and Atypical Cores.</a> Am J Hum. , (2012).</li><li>Wooddell, C. I., 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=Use+of+Evans+blue+dye+to+compare+limb+muscles+in+exercised+young+and+old+mdx+mice.">Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.</a> Muscle Nerve. 41, 487-499 (2010).</li><li>Hall, T. E., 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=The+zebrafish+candyfloss+mutant+implicates+extracellular+matrix+adhesion+failure+in+laminin+alpha2-deficient+congenital+muscular+dystrophy.">The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin alpha2-deficient congenital muscular dystrophy.</a> Proc Natl Acad Sci U S A. 104, 7092-7097 (2007).</li></ol>

Les auteurs ont aucun intérêt financier concurrentes ou d&#39;autres conflits d&#39;intérêts.

Poisson zèbre sont en train de devenir un outil puissant pour l&#39;étude de 2,29 de maladie neuromusculaire. À ce jour, le système poisson-zèbre a été utilisé pour valider le nouveau muscle mutations pathogènes 16,17,30, d&#39;élucider de nouveaux mécanismes pathologiques 18, et d&#39;identifier de nouveaux médicaments potentiellement thérapeutiques 12,24. Ces efforts collectifs ont établi l&#39;utilité du poisson zèbre pour modéliser les maladies neuromusculaires humaines. Cependant, malgré les progrès réalisés avec des modèles de poisson zèbre et de mammifères, il ya des options de traitement limitées pour les patients dans le large spectre de maladies neuromusculaires. Par conséquent, une forte demande existe pour le développement de la thérapie pour ce groupe de maladies dévastatrices. Parallèlement à cette demande de la thérapeutique est la nécessité correspondante de l&#39;innovation expérimentale en cours, ainsi que l&#39;analyse rigoureuse afin de vérifier de nouveaux modèles animaux et des stratégies thérapeutiques putatifs. Analyse EBD est couramment utilisé dans les modèles de souris dele tissu de l&#39;étude et des dommages cellulaires dans le cerveau, le cœur, et squelettique 27,31 musculaire. Plus particulièrement, EBD est largement utilisé dans des modèles murins de divers sous-types de dystrophie musculaire pour montrer la gravité de la membrane musculaire instabilité et endommager 8. L&#39;utilisation d&#39;EBD pour révéler dommages à la membrane musculaire est un paramètre de soutien à établir des similitudes du modèle animal à l&#39;état de la maladie humaine 9. La puissance de EBD chez la souris a dirigé plusieurs laboratoires, y compris notre propre, de développer et d&#39;appliquer EBD aux modèles de poisson zèbre de maladie neuromusculaire. En raison de l&#39;applicabilité de l&#39;analyse EBD, cette technique est activement mis en œuvre pour corroborer les modèles de poisson zèbre à l&#39;état de la maladie humaine 11,15,22,24,32. Les larves avec des membranes musculaires endommagées aura EBD l&#39;absorption et la fluorescence rouge par conséquent à l&#39;intérieur des fibres musculaires. Fluorescence observée dans l&#39;espace entre les fibres, mais pas à l&#39;intérieur des fibres musculaires individuelles peuvent également être informatif de fibres de détachement de la membrane basale en til absence de dommages de la membrane, fournissant des détails de diagnostic utile. Analyse EBD possède application potentielle au-delà de la validation de modèle animal. Les efforts de notre laboratoire ont récemment démontré que l&#39;analyse EBD est bénéfique dans la validation de nouveaux médicaments potentiellement thérapeutiques 24. Déterminer si les traitements thérapeutiques potentiels réduire ou supprimer EBD absorption dans les modèles de maladies neuromusculaires peut signifier action thérapeutique pertinente 8. Ce type d&#39;analyse peut aider à établir le mécanisme (s) de la thérapeutique et élargit l&#39;application de l&#39;analyse EBD. Comme avec de nombreuses techniques, l&#39;analyse n&#39;a EBD plusieurs mises en garde doivent être observées lors de la conception et de la pratique expérimentale. Par exemple, il peut être difficile d&#39;identifier la raison de la CCV épaississement du tissu avec l&#39;âge. En outre, il est facile d&#39;endommager les larves dans la préparation avant et pendant l&#39;injection péricardique, réduisant chiffres expérimentaux et l&#39;augmentation du besoin pour préparer un grand nombre de larves.En outre, les dégâts physiques que les larves lors de la manipulation et de l&#39;injection peut entraîner des faux positifs comme le muscle endommagé peut prendre jusqu&#39;à EBD. Afin de surmonter certains de ces obstacles, nous avons décrit une stratégie de co-injection dans cet article vidéo qui permet l&#39;identification fiable et facile des larves avec succès infusion de colorant immédiatement après injection et avant l&#39;analyse ultérieure. Le FITC-dextran contrôles de co-injection pour l&#39;injection de succès, en permettant de EBD confirmation dans le système vasculaire avant son absorption par les fibres musculaires. Cela peut être particulièrement utile comme EBD fluorescence devient très diffus dans les larves après plusieurs heures sinon recueillis dans les fibres musculaires; en tant que tel, il peut être difficile à détecter. En outre, à côté de la CCV et EBD injection dans la cavité du corps jaune ou peut, après incubation, la fluorescence rouge entraîner diffus semblable à contrôler embryons, mais avec le risque réduit d&#39;absorption par les fibres musculaires endommagées. Collectivement, ces grotteats suggèrent injection EBD exige de la patience et de la pratique afin de parvenir à des résultats cohérents et fiables. En tout, nous décrivons une méthode pratique et simple d&#39;effectuer des analyses sur EBD larves de poisson zèbre. À ce jour, l&#39;utilisation du poisson zèbre comme un système modèle, en particulier comme un modèle de la maladie humaine, a connu une expansion rapide. Cette expansion est due en partie à la poursuite du développement et la modification des techniques expérimentales qui améliorent les avantages sur le système actuel de poisson zèbre. La technique d&#39;injection EBD fournit un outil supplémentaire et puissant à l&#39;arsenal d&#39;un chercheur pour la validation et l&#39;étude des modèles de maladies du muscle de poisson zèbre. La mise en œuvre continue et la modification de cette technique a le potentiel d&#39;aider à découvrir de nouvelles stratégies thérapeutiques ainsi que les mécanismes de la maladie causant.

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.

Le mélange d&#39;injection EBD a été injecté dans la CCV de mutants homozygotes sapje frères et sœurs de type sauvage à 3 DPF. Les injections qui ont rempli les cavités cardiaques (figure 1B) ont ensuite été analysées pour l&#39;injection avec succès par la visualisation de FITC-dextran dans le système vasculaire sous fluorescence verte (figure 2). Après une période d&#39;incubation de 4 heures, EBD absorption a été examinée au niveau des somites utilisant la microscopie à fluorescence. Frères et sœurs de type sauvage ne ​​présentaient pas de fluorescence à l&#39;intérieur de EBD des fibres musculaires visibles (Figure 3A), tandis que les mutants homozygotes sapje EBD ont montré l&#39;absorption, ce qui indique des dommages à la membrane musculaire 15 (figure 3B). <img alt="Figure 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> Figure 1. Injecter EBD mélange d&#39;injection dans la veine cardinale commune (CCV) d&#39;un poisson zèbre Embryo. Embryon (A) non injectée. La flèche indique l&#39;emplacement idéal pour l&#39;injection de CCV. (B) d&#39;injection réussie dans la CCV. Le colorant pénètre dans les cavités cardiaques (flèche) et commence à être pompé à travers le système vasculaire. (C) Une injection de CCV échec se traduira par une partie ou tout le colorant entrant dans le sac vitellin de l&#39;embryon (flèche). <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" src="/files/ftp_upload/53183/53183fig2.jpg" /> Figure 2. Les embryons peuvent être triés pour injection réussie en observant la distribution de FITC-dextran sous fluorescence verte à travers le système vasculaire immédiatement après l&#39;injection et avant EBD absorption.S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure. <img alt="Figure 3" src="/files/ftp_upload/53183/53183fig3.jpg" /> Figure 3: EBD sera repris par fibres avec des membranes endommagées fratrie (A) de type sauvage ne ​​présentant pas de fluorescence EBD dans les fibres musculaires.. (B) Sapje ​​mutant homozygote avec EBD fluorescence à l&#39;intérieur des fibres musculaires multiples (flèches). Toutes les larves ont été injectés avec le mélange d&#39;injection EBD et analysée après une période d&#39;incubation de 4 heures à 3 dpf. Frères et sœurs et mutants ont été classés par le détachement de la fibre musculaire avant l&#39;injection de CCV. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</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 des Muscle intégrité poisson zèbre larves squelettique avec colorant bleu Evans

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 vues.  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.

Vidéo: 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.

Capture réparation des tissus chez le poisson zèbre larves avec Time-lapse Brightfield stéréomicroscopie

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 ...

Recherche

Biologie du développement

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.

Induction de aiguë Skeletal Muscle Régénération par cardiotoxine 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.

Microstructurées dispositifs pour Microinjection optimisée et l’imagerie des larves de poisson zèbre

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.

Application d’une Stimulation chronique pour étudier les Rat d’induite par l’activité Contractile du Muscle squelettique phénotypique Adaptations

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.

Identification des cellules satellites musculaires squelettiques par Immunofluorescence avec des anticorps de laminine et Pax7

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.

Isolement, culture, caractérisation et différenciation des cellules de l'ancêtre musculaire humain de la procédure de biopsie musculaire squelettique

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.

Exécution des xénogreffes de muscle squelettique humain dans les souris immunodéficientes

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.

Isolement et différenciation des myoblastes primaires des explantations de muscle squelettique de souris

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.

Immunohistochimie du mont entier dans les embryons et larves de poissons-zèbres

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.

Examen de la régénération musculaire dans les modèles de poisson-zèbre de maladie musculaire

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