Damos las gracias a Bob Nowak (Andor Technology) para la ayuda técnica y asesoramiento. SA-S. con el apoyo de una beca Heisenberg de la Deutsche Forschungsgemeinschaft (DFG). Este trabajo fue apoyado por la DFG subvención SE2016/7-1.

1. Etiquetado de células individuales Inyectar una célula de embriones etapa con un plásmido que codifica GFP o cualquier proteína GFP-fusión bajo el control de un promotor de β-actina. Durante el desarrollo, la GFP se expresa entonces en forma de mosaicos. Aquí, se utilizó el constructo transgénico Tg [β-actina: α-actinina-GFP], que coloca la proteína de fusión α-actinina-GFP bajo el control del promotor de la β-actina. 2. Embriones Embedding <ol class="lalpha"><li> Recoger embriones de pez cebra y mantener en condiciones normales hasta 28 HPF (puesta en escena de acuerdo a 12). </li><li> Embriones Dechorionate bajo el microscopio estereoscópico con fórceps (Dumont # 5). </li><li> Preparar un portaobjetos de microscopio con un anillo de vaselina. </li><li> Preparar un 1% de bajo punto de fusión de agarosa / 10% de solución de tricaína con medio E3: hervir 100 mg de agarosa en 10 ml de solución de E3 para obtener un líquido homogéneo y solución de agarosa; alícuota y tiendasin utilizar una solución de agarosa a -20 ° C para su uso posterior. Para uso inmediato, dejar que la solución se enfríe hervida y mantener en el máximo. 39 ° C. Añadir la solución de stock de tricaína para obtener una concentración final de 10% tricaína. Tanto tricaína y agarosa son necesarias para evitar los movimientos embrionarios. </li><li> Insertar un solo embrión en 1% de agarosa de bajo punto de fusión / 10% tricaína / E3 medio dentro del anillo de la jalea de petróleo, con el embrión por el que se forma natural en el lado; suavemente un cubreobjetos, sólo para mantener el embrión en su lugar y para evitar un convexo superficie que se rompería la luz. Idealmente, el tejido muscular embrionario que se dirigirá debe tocar el cubreobjetos. </li></ol> 3. Embriones láser heridas <ol class="lalpha"><li> Preparar el láser micropoint y el microscopio: <ol class="lroman"><li> Por favor, consulte el manual de micropoint láser para obtener instrucciones detalladas sobre cómo configurar y utilizar el láser en conjunción con el microscopio. </li><li> Seleccionar la adecuadacolorante para la generación de un haz de láser 435 nm de longitud de onda. </li><li> Ajuste la potencia del láser mediante el control deslizante de atenuación. La potencia del láser debe ser lo suficientemente fuerte como para romper una superficie de vidrio con un solo pulso (por ejemplo, el enfoque sobre el cubreobjetos de la muestra para probar esto). Esta es la potencia de láser requerida para la ablación celular. </li></ol></li><li> Centrarse en la región o célula de interés utilizando la retícula instalada en el ocular del microscopio. Utilice un objetivo de 20x (40x permitirá daños más precisa, pero a menudo la distancia de trabajo no es suficiente para este objetivo). </li></ol> Nota 1: El láser no sólo se lesionan las células dentro del plano de enfoque, pero también células por encima y por debajo que están dentro del haz de láser. Asegúrese de que antes del experimento que el láser está óptimamente configurado y ajustado para un enfoque alcanza su máximo en el eje z. Lo mejor es determinar la profundidad de la lesión con láser antes de realizar el experimento con el fin de saber qué células exactamente se lesionó. <ol class="lalpha" start="3"><li> Configure la luz necesaria para visualizar el experimento: campo claro para el uso normal, o si la orientación de fluorescencia de una célula fluorescente etiquetados. </li><li> Enviar un pulso de luz láser a la región objetivo, o varios impulsos, dependiendo del grado deseado de lesión. </li></ol> 4. Análisis de las heridas y la recuperación <ol class="lalpha"><li> Si se desea, el procedimiento de lesión láser puede ser grabado en vivo (por ejemplo, utilizando la opción de adquisición corriente del software Metamorph). 6 </li><li> Inmediatamente después de la herida, el embrión debe ser removido suavemente de la agarosa con pinzas y se coloca de nuevo en agua para la recuperación de huevo. </li></ol> Nota 2: La actividad muscular (natación y contracciones) es especialmente importante para la recuperación del músculo esquelético. Dado que el embrión no puede moverse debido a la exposición tricaína y la incrustación de agarosa rígido, no es RECOMEnded para mantener los embriones de un portaobjetos y un cubreobjetos durante demasiado tiempo. <ol class="lalpha" start="3"><li> Después de la recuperación, el embrión puede ser fijada y se analizaron como se desee por la luz, microscopía de fluorescencia confocal o-como se indica. </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>

No hay conflictos de interés declarado.

Laser lesión mediada es un método poderoso para infligir heridas de un tamaño deseado mediante la ablación de las células con el fin de estudiar la regeneración bajo condiciones controladas en el embrión de pez cebra. En particular, las células pueden ser dirigidos con precisión (Figura 2) y tanto el área de la lesión así como de temporización puede ser controlada. Posteriormente, el sitio de la lesión y los procesos de regeneración son fácilmente controlados, registrados (Figura...

<table border="1"><tbody><tr><td> Calefacción bloque </td><td></td><td></td><td></td></tr><tr><td> Par N º 5 fórceps </td><td> Dumont </td><td></td><td></td></tr><tr><td> Portaobjetos de vidrio </td><td> Menzel </td><td></td><td> 76 x 26 mm </td></tr><tr><td> Cubreobjetos </td><td> Roth </td><td></td><td> 50 x 24 mm # 1 </td></tr><tr><td> Jalea de petróleo </td><td></td><td></td><td></td></tr><tr><td> Estereomicroscopio </td><td> Leica </td><td></td><td> MZFLIII </td></tr><tr><td> Micropoint láser </td><td> Andor Tecnología </td><td></td><td></td></tr><tr><td> Microscopio de fluorescencia </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> El software Metamorph </td><td> Molecular Devices </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Reactivos <ul><li> Bajo punto de fusión de agarosa (# 50081,Lonza) </li><li> Tricaína solución madre: 400 mg tricaína (# A-5040, Sigma-Aldrich) / 100 ml dH 2 O pH 9,0 </li><li> Medio E3 (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

Varios enfoques experimentales se han utilizado en ratón para inducir lesión muscular con el objetivo de estudiar la regeneración muscular, incluyendo inyecciones myotoxin (bupivacaína, cardiotoxina o notexin), trasplantes musculares (denervación-desvascularización regeneración inducida), el ejercicio intenso, sino también modelos murinos de distrofia muscular como el ratón mdx (para una revisión de estos enfoques ver 1). En el pez cebra, los enfoques genéticos incluyen mutantes que exhiben fenotipos de distrofia muscular (como runzel 2 o sapje 3) y antisentido morfolinos de oligonucleótidos que bloquean la expresión de genes asociados a la distrofia 4. Además, los enfoques químicos también son posibles, por ejemplo, con la galantamina, un compuesto químico inhibiendo la acetilcolinesterasa, resultando así en hipercontracción, que finalmente conduce a la distrofia muscular 5. Sin embargo, los enfoques genéticos y farmacológicos generalmente afectar a todos los músculos dentro de un individuo, mientras que la extensión de las lesiones infligidas físicamente son más fácilmente controlados espacial y temporalmente 1. Lesión física localizada permite la evaluación de músculo contralateral como control interno. De hecho, recientemente hemos utilizado láser mediada por ablación celular para estudiar la regeneración del músculo esquelético en el embrión de pez cebra 6, mientras que otro grupo informó recientemente el uso de un láser de dos fotones (822 nm) a dañar muy localmente la membrana plasmática de músculo individual embrionario de pez cebra células 7. Aquí, se presenta un método para utilizar el láser micropoint (Andor Technology) para lesión celular del músculo esquelético en el embrión de pez cebra. El láser micropoint es un láser de alta energía, que es adecuado para la ablación de células reconocidas en una longitud de onda de 435 nm. El láser está conectado a un microscopio (en nuestra configuración, un microscopio óptico de Zeiss) de tal manera que el microscopio se puede utilizar en el mismo tiempo fo enfocar la luz láser sobre la muestra y para la visualización de los efectos de la herida (campo claro o fluorescencia). Los parámetros para el control de pulsos de láser incluyen longitud de onda, intensidad, y número de pulsos. Debido a su transparencia y el desarrollo embrionario externo, el embrión de pez cebra es altamente susceptible, tanto para la lesión inducida por láser y para el estudio de la recuperación posterior. Entre 1 y 2 días después de la fecundación, las células del músculo esquelético somitic someterse a la maduración progresiva de anterior a posterior debido a la progresión de somitogenesis desde el tronco hasta la cola 8, 9. En estas etapas, los embriones espontáneamente crispar e iniciar la natación. El pez cebra se ha reconocido recientemente como un organismo vertebrado importante modelo para el estudio de la regeneración de los tejidos, como muchos tipos de tejidos (cardiacas, neuronal, vascular, etc) se puede regenerar después de la lesión en el adulto pez cebra 10, 11.

Laser lesión mediada se realizó en inmovilizadas 1 día de edad embriones. Como se muestra en la Figura 1, un láser de pulsos pocos puede generar una pequeña herida, fácilmente reconocible por los dañados, enrollados, actina ricos en miofibrillas que normalmente se extendía entre los límites somite. Un mayor número de pulsos de láser sin embargo resultará en un bloque de somite dañado masivamente, en donde la mayoría de las miofibrillas son destruidos. No obstante, se puede observar que, li...

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

El método que aquí se presenta incluye el daño exacto de embriones de pez cebra en vivo con pulsos láser de alta energía y el posterior análisis de estas lesiones y su recuperación con el tiempo. También mostramos cómo genéticamente etiquetado individual o grupos de células del músculo esquelético se puede seguir durante y después de la luz láser daño inducido.

Laser-infligido lesiones de músculo esquelético embrionario de pez cebra

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 vistas.  Max Delbrück Center for Molecular Medicine. El método que aquí se presenta incluye el daño exacto de embriones de pez cebra en vivo con pulsos láser de alta energía y el posterior análisis de estas lesiones y su recuperación con el tiempo. También mostramos cómo genéticamente etiquetado individual o grupos de células del músculo esquelético se puede seguir durante y después de la luz láser daño inducido.

Video: 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.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

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

Investigación

Biología

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.

Aislamiento mitocondrial del músculo esquelético

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.

Purificación de los progenitores del músculo esquelético

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.

Análisis de embriones y larvas de pez cebra esqueléticas miofibras de Preparativos disociadas

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.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

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.

Electroporación del ADN plásmido en el músculo esquelético del ratón

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-respirometría de alta resolución del músculo esquelético equino

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

Cuantificación de la infiltración grasa del músculo esquelético mediante descelularización

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 destacado: Cuantificación basada en la descelularización de la infiltración grasa del músculo esquelético  

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.