Queremos agradecer Trent Waugh para a sua assistência técnica. Reconhecemos também o Departamento de Pediatria do Hospital for Sick Children e cura da distrofia muscular congênita (DMC) para seu financiamento generoso para este projeto.

1. Preparação de Agar placas de injeção (Tempo: 45 min) <ol><li> Ferver 2% a 3% de agarose em meio E3 e permita que a solução arrefecer ligeiramente no banco. Nota: O número de placas de injecção a ser preparado dita a quantidade de agarose necessária. Cada placa de injecção necessita de cerca de 35 ml da solução de agarose. </li><li> Depois de ferver, permitir que a agarose a arrefecer até a temperatura desejada seja atingida (por exemplo., 45 ° C) de acordo com as instruções do fabricante do molde de injecção. </li><li> Pour cerca de 35 ml de agarose a arrefecido em um prato de 100 mm. </li><li> Coloque uma extremidade do molde de injeção preferido na solução, em seguida, estabelecer restante do molde sobre solução de agarose (isso vai ajudar a reduzir a ocorrência de bolhas de ar). </li><li> Permitir que a solução de agarose para solidificar quer à TA ou a 4 ° C durante aproximadamente 30 min. </li><li> Usar uma espátula para separar uma extremidade do molde a partir da agarose sólida. Lentamente remover o restante do m<html> velho. </li></ol> 2. Preparação de Evans Blue Dye (EBD) Injeção Mix (Tempo: 30 min) <ol><li> Adicione um estoque de 1% de CAE em solução de 1X Ringer (NaCl 155; KCl 5 mM; CaCl2 2 mM; MgCl2 1 mM; 2 mM de Na 2 HPO 4; HEPES 10 mM; glucose 10 mM; pH a 7,2), que pode ser armazenado à temperatura ambiente. </li><li> Adicione uma solução de reserva de isotiocianato de fluoresceína (FITC) -dextrano MW 10.000 kDa a 25 mg / ml em solução e armazenamento de 1X Ringer a -20 ° C. </li><li> Preparar a mistura de injecção através da diluição para 0,1% CAE directamente na solução estoque de estoque de FITC-dextrano (isto é, para um volume de trabalho de 100 uL finais:. Misturar 10 ul de 1% CAE em 90 ul de estoque de dextrano FITC). </li><li> Misture bem injeção vórtice (que deve ficar verde) e manter fora da luz direta envolvendo injeção de tubo de mistura em papel alumínio. </li></ol> 3. Preparação Injeção EBD (Tempo: Cerca de 30 min) ent &quot;&gt; Nota: protocolo funciona melhor com larvas de 3-7 dias após a fertilização (DPF). <ol><li> Placa de injecção pré-aquecer a RT. </li><li> Configure aparelho de injecção, organizando micromanipulador na placa de metal e ficar ao lado do microscópio que está sendo usado para a injeção. Ligue controlador de microinjeção ar conduzido. Nota: O sistema de injeção preferido irá variar em laboratório e não deve alterar resultado da análise. </li><li> Voltar encher agulha de injecção com cerca de 2-4 ul da mistura EBD. </li><li> Calibrar volume de injeção de cerca de 5 nl de mix EBD. Nota: Injecção de calibragem de volume dependerá método de calibração. Uma injecção de êmbolo accionado directamente pode ser definido para um determinado volume de injecção ao passo que os injectores de gás de pressão terá o volume de injecção de bolus por via do volume calibrado com a utilização de um micrómetro. </li><li> Placa de injecção molhada com solução de Ringer 1X e remover o excesso de poços. </li><li> Larvas pré-prazer, com 0,04% de acetato de 3-aminobenzoato de sal metanossulfonatot (tricaina) diluídos em solução de Ringer 1X para imobilizar larvas antes do início da injecção. Nota: Garantir larvas são completamente immotile é importante como uma injecção adequada é difícil com qualquer movimento residual. </li><li> Coloque larvas anestesiados nos poços das placas de agar utilizando injecção de uma pipeta de vidro. Certifique-se de que as larvas são completamente dentro do poço e deitado sobre seu lado. Nota: O número de larvas por poço é até o experimentador. </li><li> Depois de larvas são colocadas nas cavidades, remover excesso de solução de Ringer para minimizar o movimento larvas dentro do poço. Deixar uma quantidade residual de solução para que as larvas não desidratar. </li></ol> 4. Injeção Pericárdica de Zebrafish larvas com EBD (Tempo: Dependente do número de larvas de injecção, estima 1-3 hr) <ol><li> Colocar a placa de injecção contendo as larvas em um escopo de dissecação onde as injecções serão realizados. </li><li> Posicione a agulha pipeta de injeção contendoCAE misturar mais de um larvas do peixe. </li><li> Reposicionar a placa de injecção através da rotação de modo que a agulha de injecção está próximo do coração do larvas e cerca de 45 ° ventralmente em relação ao eixo ântero-posterior. </li><li> Inserir a agulha de injecção na veia cardinal comum (CCV) na região da veia na porção anterior da gema quando a veia está inicialmente rodar na direcção dorsal (Figura 1). Nota: A ampliação de até 40x pode ser útil para ver claramente o CCV. </li><li> Injectar 5 nl de mix EBD e manter agulha de injeção na posição para 5-8 segundos para minimizar a perda imediata do mix EBD. Nota: Uma boa injecção terá corante coloração visto nas câmaras do coração (Figura 1). Se EBD mistura não é observada no coração e, em seguida injectando um adicional de 5 nl mistura EBD pode ser suficiente para induzir a absorção do corante. Alternativamente, o embrião pode ser descartado. Nota: Em algumas situações, o coração pode parar de bater. Se este óccurs, continuar a acompanhar as larvas de 20-40 seg. Tipicamente, o coração recomeça a bater como o corante se move através do sistema circulatório. </li><li> Passar para a próxima larvas e repita. </li><li> Identificar embriões injectados com sucesso mediante a observação da presença de FITC-dextrano na vasculatura imediatamente após a injecção (Figura 2). </li></ol> 5. Incubação e EBD Captação (Tempo: 4-6 hr) <ol><li> Após o número desejado de larvas são injectados, larvas injectadas retorno para a solução de Ringer 1X sem tricaina em placas de 100 mm. </li><li> Mantenha pratos embrulhados em papel alumínio. Nota: Manter larvas injetadas no escuro aumenta significativamente as taxas de sobrevivência e garante a maior consistência na intensidade do sinal. Embalagem em folha de alumínio é especialmente importante durante o período de tempo que as larvas estão fora da incubadora. </li><li> Permitir que as larvas incubar a 28,5 ° C durante 4-6 horas para assegurar a aceitação EBD suficiente. </li></ol&gt; 6. Visualização da EBD no músculo (Tempo: Dependente do número de larvas e de injecção tipo de microscopia, estimados 0,5-3 hr) <ol><li> Antes de imagiologia, anestesiar as larvas com 0,04% tricaina para impedir o movimento. </li><li> Ver larvas sob fluorescência vermelha para determinar se está a ocorrer EBD captação no músculo esquelético (Figura 3). </li></ol>

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Os autores não têm concorrentes interesses financeiros ou outros conflitos de interesse.

Zebrafish estão emergindo como uma poderosa ferramenta para o estudo de 2,29 doença neuromuscular. Até à data, o sistema de peixe-zebra foi utilizado para validar causadores de doenças musculares nova mutações 16,17,30, elucidar novos patomecanismos 18, e potencialmente identificar novas drogas terapêuticas 12,24. Estes esforços coletivos estabeleceram a utilidade do peixe-zebra para modelar doenças neuromusculares humanas. No entanto, apesar dos avanços feitos com modelos de peixe-zebra e mamíferos, há opções limitadas de tratamento para pacientes dentro do amplo espectro de condições neuromusculares. Portanto, existe uma grande demanda para o desenvolvimento de terapia para esse grupo de doenças devastadoras. Em paralelo a essa demanda por terapêutica é a correspondente necessidade de inovação experimental em curso, bem como uma análise rigorosa para verificar novos modelos animais e estratégias terapêuticas putativos. Análise EBD é comumente usado em modelos de ratos paratecido estudo e danos celulares no cérebro, coração, músculo esquelético e 27,31. Mais notavelmente, EBD é amplamente utilizado em modelos de ratos com vários subtipos de distrofia muscular para mostrar a gravidade da membrana muscular instabilidade e danificar 8. O uso de CAE para revelar danos membrana muscular é um parâmetro de suporte que estabelece semelhanças entre o modelo animal para o estado de doença humana 9. O poder da EBD no rato levou vários laboratórios, inclusive o nosso, para desenvolver e aplicar EBD para modelos de peixe-zebra de doença neuromuscular. Devido à aplicabilidade da análise EBD, esta técnica está ativamente sendo implementadas para corroborar os modelos de peixe-zebra para o estado de doença humana 11,15,22,24,32. Larvas com as membranas musculares danificadas terá captação EBD e fluorescência vermelha, portanto, dentro de fibras musculares. A fluorescência observada no espaço entre as fibras, mas não nas fibras musculares individuais podem também ser informativo de fibras destacados do porão em t membranaele ausência de dano à membrana, fornecendo detalhes úteis de diagnóstico. Análise EBD tem potencial de aplicação para além validação do modelo animal. Os esforços do nosso laboratório demonstraram recentemente que a análise EBD é benéfica na validação de medicamentos terapêuticos potencialmente novos 24. Determinar se os potenciais tratamentos terapêuticos reduzir ou abolir a captação EBD em modelos de doenças neuromusculares podem significar ação terapêutica relevante 8. Este tipo de análise pode ajudar a estabelecer o mecanismo (s) de terapêuticas e expande a aplicação da análise EBD. Tal como acontece com muitas técnicas, análise EBD tem muitos cuidados a serem observados durante a concepção e prática experimental. Por exemplo, pode ser um desafio para identificar o CCV devido ao espessamento do tecido com a idade. Além disso, é fácil de danificar larvas em preparação antes e durante a injecção do pericárdio, reduzindo a contagem experimentais e aumentando a necessidade de preparar um grande número de larvas.Além disso, os danos físicos causados ​​ao larvas durante o manuseio e de injecção pode resultar em falsos positivos como músculo danificado pode levar até EBD. A fim de ultrapassar alguns destes obstáculos, descrevemos uma estratégia de co-injecção neste artigo de vídeo que permite a identificação fácil e fiável das larvas com infusão de corante sucesso imediatamente após a injecção e antes da análise subsequente. O FITC-dextrano controlos co-injecção para a injecção bem sucedido, permitindo a confirmação de CAE na vasculatura antes da sua absorção pelas fibras musculares. Isto pode ser particularmente útil como EBD fluorescência torna-se altamente difusa em larvas após várias horas, se não recolhidos nas fibras musculares; como tal, pode ser difícil de detectar. Além disso, a falta de CCV e injectando EBD na gema ou cavidade corporal pode, após a incubação, resultam em fluorescência vermelha difusa semelhante para controlar embriões, ainda com a probabilidade reduzida de absorção pelas fibras musculares danificadas. Coletivamente, estas cavernaats sugerem injeção EBD requer paciência e prática para alcançar resultados consistentes e confiáveis. Ao todo, nós descrevemos um método prático e simples para realizar a análise EBD em larvas do peixe. Até à data, a utilização de peixe-zebra como um sistema modelo, especialmente como um modelo da doença humana, tem vindo a expandir-se rapidamente. Esta expansão é parcialmente devido ao contínuo desenvolvimento e modificação de técnicas experimentais que melhoram sobre as vantagens atuais do sistema peixe-zebra. A técnica de injeção EBD fornece uma ferramenta adicional e poderosa para arsenal do pesquisador para a validação e estudo de modelos de doenças músculo de peixe-zebra. A implementação em curso e modificação desta técnica tem o potencial de ajudar a descobrir novas estratégias terapêuticas, bem como mecanismos que causam doenças.

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.

A mistura de injecção EBD foi injectada na CCV de mutantes homozigóticos sapje e irmãos de tipo selvagem no 3 dpf. As injecções que encheu as câmaras do coração (Figura 1B) foram então analisadas por injecção bem sucedida por meio da visualização de FITC-dextrano na vasculatura sob fluorescência verde (Figura 2). Após um período de incubação de 4 h, EBD absorção foi examinada a nível somito utilizando microscopia de fluorescência. Irmãos de tipo selvagem exibiram nenhuma fluorescência EBD dentro de quaisquer fibras musculares visíveis (Figura 3A), ao passo que os mutantes homozigóticos sapje EBD mostrou absorção, indicando os danos para a membrana muscular 15 (Figura 3B). <img alt="figura 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> Figura 1. Injectar EBD mix injeção na veia cardinal comum (CCV) de um peixe-zebra embryo. Embrião (A) não injectado. Seta indica a localização ideal para a injeção CCV. (B) injecção bem sucedido para a CCV. O corante entra nas câmaras do coração (seta) e começa a ser bombeado através da vasculatura. (C) Uma injeção vencida CCV irá resultar em alguns ou todos do corante entrar no saco vitelino do embrião (seta). <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53183/53183fig2.jpg" /> Figura 2. Os embriões podem ser classificadas para injeção de sucesso, observando distribuição FITC-dextrano sob fluorescência verde em toda a vasculatura imediatamente a seguir à injecção e antes da EBD captação.Por favor clique aqui para ver uma versão maior desta figura. <img alt="Figura 3" src="/files/ftp_upload/53183/53183fig3.jpg" /> Figura 3: EBD será retomada por fibras com membranas danificadas irmãos (A) do tipo selvagem mostrando ausência de fluorescência EBD nas fibras musculares.. (B) Sapje ​​mutante homozigótica com fluorescência EBD dentro múltiplas fibras musculares (setas). Todas as larvas foram injectados com a mistura de injecção CAE e analisados ​​após um período de incubação de 4 horas a 3 dpf. Irmãos e mutantes foram ordenados por fibra muscular destacamento antes da injeção CCV. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</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.

Análise de Zebrafish larvas Músculo Esquelético Integrity com 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 ...

analysis-zebrafish-larvae-skeletal-muscle-integrity-with-evans-blue

Research

JoVE Journal

Developmental Biology

9.0K visualizações.  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.

Vídeo: 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.

Captura de Reparo de Tecidos em Zebrafish larvas com a Time-lapse Brightfield lupa estereoscópica

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 ...

Biologia do Desenvolvimento

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.

Indução da Aguda Skeletal regeneração muscular por cardiotoxina 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.

Microstructured dispositivos para Microinjection otimizado e imagem latente de larvas de Zebrafish

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.

Aplicação da estimulação crônica para estudar ratos induzida pela atividade contrátil adaptações fenotípica de músculo esquelético

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.

Identificação de células satélite de músculo esquelético por imunofluorescência com anticorpos de laminina e 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.

Isolamento, cultura, caracterização e diferenciação de células progenitoras do músculo humano do procedimento de biópsia do músculo esquelético

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.

Realizando xenoenxertos de músculo esquelético humano em camundongos imunodeficientes

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.

Isolamento e diferenciação de Mioblasts primários de explantes do músculo esquelético do rato

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.

Imunohistoquímica do Monte Inteiro em Embriões e Larvas de Zebrafish

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.

Examinando a regeneração muscular em modelos de zebrafish de doença muscular

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