Agradecemos Bob Nowak (Andor Technology) para obter ajuda e aconselhamento técnico. SA-S. é apoiado por uma bolsa de Heisenberg da Deutsche Forschungsgemeinschaft (DFG). Este trabalho foi financiado pelo DFG concessão SE2016/7-1.

1. Rotular Células individuais Injectar embriões unicelulares palco com um plasmídeo que codifica GFP ou de qualquer proteína de fusão GFP sob o controlo de um promotor β-Actina. Durante o desenvolvimento, a GFP é então expressa de uma forma de mosaico. Aqui, utilizou-se o construto transgénico Tg [β-actina: α-actinina-GFP], o que coloca a proteína de fusão α-actinina-GFP sob o controlo do promotor da β-actina. 2. Incorporação de embrião <ol class="lalpha"><li> Coletar embriões zebrafish e manter em condições normais até 28 hpf (estadiamento de acordo com a 12). </li><li> Embriões Dechorionate sob o estereomicroscópio com uma pinça (Dumont # 5). </li><li> Prepara-se uma lâmina de microscópio com um anel de vaselina. </li><li> Preparar um 1% de baixo ponto de fusão de agarose / 10% de solução de tricaina com meio E3: fervura 100 mg de agarose em 10 ml de solução E3 para obter um líquido homogéneo e solução de agarose; aliquota e armazenarsolução de agarose não utilizado a -20 º C para posterior utilização. Para uso imediato, deixe a solução fervida esfriar e manter no máximo. 39 ° C. Adicionar tricaina solução stock para se obter uma concentração final de 10% tricaina. Ambos tricaina e agarose são necessárias para evitar movimentos embrionários. </li><li> Incorporação de um único embrião de 1% de ponto de fusão baixo a médio de agarose / 10% tricaina / E3 dentro do anel de vaselina, com o embrião fixa naturalmente no lado; suavemente cobrir com uma lamela apenas para manter o embrião no lugar e evitar um convexo superfície que iria quebrar a luz. Idealmente, o tecido do músculo embrionário que serão alvo deve tocar na lamela. </li></ol> 3. Embryo Laser ferimento <ol class="lalpha"><li> Prepare o laser Micropoint e do microscópio: <ol class="lroman"><li> Por favor, consulte o manual do laser Micropoint para instruções detalhadas sobre como configurar e utilizar o laser em conjunto com o microscópio. </li><li> Selecione o corretocorante para a geração de um feixe de laser 435 nm de comprimento de onda. </li><li> Ajustar a potência do laser usando o controle deslizante de atenuação. A potência do laser deve ser suficientemente forte para quebrar a superfície de vidro com um único impulso (por exemplo, o foco sobre a lamela de sua amostra para testar esta). Esta é a potência do laser necessária para a ablação de células. </li></ol></li><li> Concentre-se na região ou célula de interesse, utilizando o retículo instalado na ocular do microscópio. Use uma objetiva de 20x (40x permita danos mais preciso, mas muitas vezes a distância de trabalho não é suficiente para essa lente). </li></ol> Nota 1: O laser não só danificam as células no interior do plano de focagem, mas também as células acima e abaixo que se encontram dentro do raio laser. Certifique-se antes da experiência que o laser é perfeitamente configurado e ajustado para um foco máxima dentro do eixo z. É melhor para determinar a profundidade da lesão do laser antes de realizar o ensaio, a fim de saber qual células exatamente será ferido. <ol class="lalpha" start="3"><li> Defina-se a luz necessária para a visualização do experimento: brightfield para uso normal, ou fluorescência, se uma célula alvo marcada com fluorescência. </li><li> Enviar um impulso de luz laser para a região alvo, ou em vários pulsos, dependendo do grau desejado de lesão. </li></ol> 4. Análise de ferimento e Recuperação <ol class="lalpha"><li> Se desejado, o procedimento de lesão do laser pode ser gravado (por exemplo, utilizando a opção de aquisição fluxo do software Metamorph) 6. </li><li> Imediatamente após o ferimento, o embrião deve ser removido suavemente a partir da agarose com uma pinça e colocado de novo em água de ovo para a recuperação. </li></ol> Nota 2: A atividade muscular (natação e espasmos) é especialmente importante para a recuperação do músculo esquelético. Uma vez que o embrião não pode mover-se devido à exposição tricaina e a incorporação de agarose rígida, não é RECOMEnded para manter os embriões entre lâmina e lamínula por muito tempo. <ol class="lalpha" start="3"><li> Depois da recuperação, o embrião pode ser fixado e analisadas, conforme desejado pela luz, microscopia de fluorescência confocal ou conforme indicado. </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>

Não há conflitos de interesse declarados.

Laser mediada lesão é um método poderoso para infligir feridas de um tamanho desejado, a ablação de células, a fim de estudar a regeneração em condições controladas, o embrião do peixe-zebra. Notavelmente, as células podem ser direcionados precisamente (Figura 2), e tanto a área de lesão, assim como de temporização pode ser controlada. Subsequentemente, o local da lesão e os processos de regeneração são facilmente monitorizados, registada (Figura 3) e analisadas <str...

<table border="1"><tbody><tr><td> Bloco de aquecimento </td><td></td><td></td><td></td></tr><tr><td> Par # 5 fórceps </td><td> Dumont </td><td></td><td></td></tr><tr><td> Lâminas de vidro </td><td> Menzel </td><td></td><td> 76 x 26 mm </td></tr><tr><td> Lamelas </td><td> Roth </td><td></td><td> 50 x 24 mm # 1 </td></tr><tr><td> Vaselina </td><td></td><td></td><td></td></tr><tr><td> Estereomicroscópio </td><td> Leica </td><td></td><td> MZFLIII </td></tr><tr><td> Micropoint a laser </td><td> Andor Tecnologia </td><td></td><td></td></tr><tr><td> Microscópio de fluorescência </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph software </td><td> Dispositivos Moleculares </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Reagentes <ul><li> Baixo ponto de fusão agarose (# 50081,Lonza) </li><li> Tricaina solução mãe: 400 mg tricaina (# A-5040, Sigma-Aldrich) / 100 ml de dH2O, pH 9,0 </li><li> Meio E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl2, 0,33 mM MgSO4) </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

Várias abordagens experimentais têm sido utilizados no mouse para induzir lesão muscular com o objetivo de estudar a regeneração muscular, incluindo injeções miotoxina (bupivacaína, cardiotoxina ou notexin), transplantes musculares (denervação desvascularização-regeneração induzida), exercício intensivo, mas também modelos murinos de distrofia muscular como o camundongo mdx (para uma revisão dessas abordagens ver 1). No peixe-zebra, abordagens genéticas incluem mutantes que exibem fenótipos de distrofia muscular (como runzel 2 ou sapje 3) e anti-sentido morpholinos oligonucleotídicas que bloqueiam a expressão de genes associados a distrofia 4. Além disso, as abordagens químicas também são possíveis, por exemplo, com a galantamina, um composto químico inibe a acetilcolinesterase, resultando assim em hipercontração, o que eventualmente leva a distrofia muscular 5. Abordagens entanto, genéticos e farmacológicos generally afectam todos os músculos no interior de um indivíduo, ao passo que a extensão dos ferimentos infligidos são fisicamente mais facilmente controlada espacialmente e temporalmente 1. Lesão física localizada permite a avaliação do músculo contralateral como controle interno. De facto, foi recentemente utilizado por laser de ablação celular mediada para estudar a regeneração do músculo esquelético no embrião do peixe-zebra 6, enquanto que um outro grupo recentemente relatada a utilização de um laser de dois fotões (822 nm) para danificar muito localmente na membrana plasmática de músculo de peixe zebra indivíduo embrionário células 7. Aqui, relatamos um método para a utilização do laser Micropoint (Andor Technology) por lesão do músculo esquelético de células no embrião do peixe-zebra. O laser Micropoint é um laser de alta energia, que é adequado para a ablação de células alvo num comprimento de onda de 435 nm. O laser é ligado a um microscópio (na nossa configuração, um microscópio óptico Zeiss partir) de tal modo que o microscópio pode ser utilizado para o mesmo tempo fou focalizar a luz de laser sobre a amostra e para a visualização dos efeitos do ferimento (campo claro ou de fluorescência). Os parâmetros para controlar o comprimento de onda de pulsos de laser incluem, a intensidade e o número de pulsos. Devido à sua transparência e desenvolvimento embrionário externo, o embrião do peixe-zebra é altamente favorável para ambos lesão induzida por laser e para estudar a recuperação posterior. Entre 1 e 2 dias após a fertilização, somíticos células do músculo esquelético progressivamente sofrerem maturação de anterior para posterior, devido à progressão da somitogénese do tronco para a cauda 8, 9. Nessas etapas, os embriões espontaneamente se contorcer e iniciar a natação. O peixe-zebra foi recentemente reconhecido como um importante organismo vertebrado modelo para o estudo da regeneração de tecidos, tal como vários tipos de tecidos (cardíaca, neuronal, vascular, etc) pode ser regenerado após a lesão no peixe-zebra adultos 10, 11.

Lesão mediada por laser foi realizada em imobilizadas um dia de idade, os embriões. Como mostrado na Figura 1, um impulso de laser que poucos podem gerar uma pequena ferida, facilmente reconhecível pelos danificadas, enroladas, Actina ricos miofibrilas, que normalmente são esticadas entre os limites somito. Um maior número de pulsos de laser vai, porém, resultar em um bloco somito maciçamente danificados, onde a maioria das miofibrilas são destruídos. No entanto, podemos observar que, literalme...

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

O método aqui apresentado compreende a lesão precisa de embriões vivos zebrafish com pulsos de laser de alta potência e a subsequente análise das lesões e sua recuperação com o tempo. Também mostramos como geneticamente marcado individual ou grupos de células do músculo esquelético pode ser monitorado durante e após o laser danos leves induzido.

Laser-infligido Lesão de Zebrafish muscular esquelético embrionário

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 visualizações.  Max Delbrück Center for Molecular Medicine. O método aqui apresentado compreende a lesão precisa de embriões vivos zebrafish com pulsos de laser de alta potência e a subsequente análise das lesões e sua recuperação com o tempo. Também mostramos como geneticamente marcado individual ou grupos de células do músculo esquelético pode ser monitorado durante e após o laser danos leves induzido.

Vídeo: Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Microdissecção de tecidos oculares Zebrafish embrionárias

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Biologia

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 e embrionárias Cultura Microexplant Músculo Esquelético e isolamento de células-tronco musculares

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

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.

Isolamento mitocondrial do 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.

Purificação de Progenitores do 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.

Análise de Desenvolvimento Embrionário e larval Zebrafish esqueléticos miofibras de Preparações dissociadas

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 microinjeção no peixe-zebra ao modelo de lesão 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.

Eletroporação do DNA plasmídeo no músculo esquelético do rato

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.

Fluor-respirometria de alta resolução do músculo esquelético equino

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

Criolesão do pedúnculo caudal em peixe-zebra adulto

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

Autor em destaque: Um modelo de criolesão para estudar a regeneração do músculo esquelético do pedúnculo caudal em peixe-zebra adulto  

This protocol describes a cryoinjury model to induce profound damage of several caudal myomeres in adult zebrafish. This method provides a new approach for studying skeletal muscle regeneration after severe loss of tissue in non-mammalian vertebrates.