Queremos agradecer a Trent Waugh por su asistencia técnica. También reconocemos el Departamento de Pediatría en el Hospital para Niños Enfermos y Cure distrofia muscular congénita (CMD) por su generosa financiación para este proyecto.

1. Preparación de placas de agar de inyección (Tiempo: 45 min) <ol><li> Hervir 2% al 3% de agarosa en medios E3 y permita que la solución se enfríe un poco en el banco. Nota: El número de placas de inyección está preparando dicta la cantidad de agarosa necesario. Cada placa de inyección necesita aproximadamente 35 ml de la solución de agarosa. </li><li> Después de hervir, deje que la agarosa se ​​enfríe hasta alcanzar la temperatura deseada (por ejemplo., 45 ° C) según las instrucciones del fabricante molde de inyección. </li><li> Verter aproximadamente 35 ml de la agarosa enfriados en un plato de 100 mm. </li><li> Coloque un extremo del molde de inyección preferido en la solución, luego se quedó resto del molde sobre solución de agarosa (esto ayudará a reducir la aparición de burbujas de aire). </li><li> Dejar que la solución de agarosa se ​​solidifique ya sea a temperatura ambiente o 4 ° C durante aproximadamente 30 min. </li><li> Use una espátula para separar un extremo del molde de la agarosa sólida. Retire lentamente el resto de la m<html> viejo. </li></ol> 2. Preparación del colorante azul de Evans (EBD) Injection Mix (Tiempo: 30 min) <ol><li> Hacer un 1% stock de EBD en 1X solución de Ringer (NaCl mM 155; KCl 5 mM; 2 mM CaCl 2; 1 mM de MgCl 2; 2 mM Na 2 HPO 4; HEPES 10 mM; glucosa 10 mM; pH a 7,2), que se puede almacenar a temperatura ambiente. </li><li> Hacer una solución de reserva de isotiocianato de fluoresceína (FITC) -dextrano MW 10.000 kDa a 25 mg / ml en solución y la tienda de 1X Ringer a -20 ° C. </li><li> Preparar la mezcla de inyección diluyendo EBD a 0,1% directamente en solución madre de Stock FITC-dextrano (es decir, para un volumen de trabajo final de 100 l:. Mezclar 10 l de 1% EBD en 90 l de FITC Stock dextrano). </li><li> Mezclar bien la inyección de vórtice (debe ponerse de color verde) y mantenerlo fuera de la luz directa envolviendo tubo de mezcla de inyección en papel de aluminio. </li></ol> Preparación 3. EBD Inyección (Tiempo: Aproximadamente 30 min) ent &quot;&gt; Nota: Protocolo funciona mejor con larvas después de la fecundación 3-7 días (DPF). <ol><li> Placa de inyección Pre-caliente para RT. </li><li> Configurar aparato de inyección mediante la organización de micromanipulador en la placa de metal y de pie al lado del microscopio que se utilizan para la inyección. Encienda el controlador de la microinyección de aire impulsado. Nota: El sistema de inyección preferida variará por el laboratorio y no debe cambiar resultado del análisis. </li><li> Volver llenar aguja de inyección con aproximadamente 2.4 l de mezcla EBD. </li><li> Calibrar volumen de inyección a aproximadamente el 5 nl de mezcla EBD. Nota: Calibración Volumen de inyección dependerá de método de calibración. Una inyección de pistón accionado se puede fijar directamente a un volumen de inyección dado mientras que los inyectores de presión de gas tendrán que el volumen de inyección calibrado a través de volumen de bolo con el uso de un micrómetro. </li><li> Placa de inyección húmedo con una solución de 1X Ringer y eliminar el exceso de los pozos. </li><li> Larvas de Pre-tratamiento con sal metanosulfonato 0,04% de acetato de 3-aminobenzoatot (tricaína) diluido en solución de Ringer 1X para inmovilizar las larvas antes del comienzo de la inyección. Nota: Asegurar larvas son completamente inmóviles es importante ya que la inyección adecuada es difícil con cualquier movimiento residual. </li><li> Coloque larvas anestesiadas en los pocillos de las placas de inyección de agar usando una pipeta de vidrio. Asegúrese de que las larvas son completamente dentro del pozo y la mentira de su lado. Nota: El número de larvas por así es hasta el experimentador. </li><li> Después de larvas se colocan en los pocillos, eliminar el exceso de solución de Ringer para minimizar el movimiento larvas dentro del pozo. Deja una cantidad residual de solución para que las larvas no deshidratar. </li></ol> 4. Inyección pericárdico de larvas de pez cebra con EBD (Tiempo: Depende de la cantidad de inyección de larvas, que se estima 1.3 h) <ol><li> Coloque la placa de inyección que contiene las larvas en un microscopio de disección, donde se llevarán a cabo las inyecciones. </li><li> Coloque la aguja pipeta de inyección que contieneel EBD mezcla sobre un larvas de pez cebra. </li><li> Vuelva a la posición de la placa de inyección girándola para que la aguja de inyección está cerca del corazón de las larvas y de aproximadamente 45 ° ventral del eje antero-posterior. </li><li> Insertar la aguja de inyección en la vena cardinal común (CCV) en la región de la vena en la porción anterior de la yema donde la vena se está convirtiendo inicialmente en la dirección dorsal (Figura 1). Nota: con un aumento de hasta 40x puede ser útil para ver claramente la CCV. </li><li> Inyectar 5 nl de mezcla EBD y mantener la aguja de inyección en la posición de 5-8 segundos para minimizar la fuga inmediata de mezcla EBD. Nota: Una buena inyección tendrá coloración colorante se observa en las cámaras del corazón (Figura 1). Si mezcla EBD no se observa en el corazón, a continuación, la inyección de un 5 nl adicional de mezcla EBD puede ser suficiente para inducir la absorción de colorante. Alternativamente, el embrión puede ser desechada. Nota: En algunas situaciones, el corazón puede dejar de latir. Si este occurs, continuará monitoreando las larvas de 20-40 seg. Típicamente, el corazón latiendo como se reanuda el colorante se mueve a través del sistema circulatorio. </li><li> Pasar a la siguiente larvas y repetir. </li><li> Identificar embriones inyectados con éxito mediante la observación de la presencia de FITC-dextrano en la vasculatura inmediatamente después de la inyección (Figura 2). </li></ol> 5. Incubación y EBD Captación (Hora: 04.06 horas) <ol><li> Después se inyecta el número deseado de larvas, larvas de retorno inyecta a 1X solución de Ringer sin tricaína en placas de 100 mm. </li><li> Mantenga los platos envueltos en papel de aluminio. Nota: Mantener larvas inyectado en la oscuridad aumenta significativamente las tasas de supervivencia y garantiza la mayor coherencia en la intensidad de la señal. Envolver en papel de aluminio es especialmente importante para el período de tiempo que las larvas se encuentran fuera de la incubadora. </li><li> Permita que las larvas de incubar a 28,5 ° C durante 4-6 horas para asegurar la suficiente absorción EBD. </li></ol&gt; 6. Visualización de EBD en el músculo (Tiempo: Depende de la cantidad de inyección de larvas y el tipo de microscopía, prevista 0,5-3 horas) <ol><li> Antes de la formación de imágenes, anestesiar a las larvas con 0,04% tricaína para evitar el movimiento. </li><li> Ver larvas bajo fluorescencia roja para determinar si EBD absorción se produce en el músculo esquelético (Figura 3). </li></ol>

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Los autores no tienen que compiten intereses financieros u otros conflictos de intereses.

El pez cebra se perfila como una herramienta poderosa para el estudio de 2,29 enfermedad neuromuscular. Hasta la fecha, el sistema de pez cebra se ha utilizado para validar que causan enfermedades nueva musculares mutaciones 16,17,30, dilucidar nuevos mecanismos patológicos 18, e identificar potencialmente nuevas drogas terapéuticas 12,24. Estos esfuerzos colectivos han establecido la utilidad del pez cebra para modelar enfermedades neuromusculares humanos. Sin embargo, a pesar de los avances realizados con modelos de pez cebra y mamíferos, hay opciones limitadas de tratamiento para los pacientes dentro del amplio espectro de condiciones neuromusculares. Por lo tanto, existe una gran demanda para el desarrollo de la terapia para este grupo de enfermedades devastadoras. Paralelamente a esta demanda para la terapéutica es la correspondiente necesidad de innovación experimental en curso, así como el análisis riguroso para verificar nuevos modelos animales y estrategias terapéuticas putativos. Análisis EBD se utiliza comúnmente en modelos de ratón detejido estudio y el daño celular en el cerebro, el corazón y el músculo esquelético 27,31. Más notablemente, EBD se utiliza ampliamente en modelos de ratón de diversos subtipos distrofia muscular para mostrar la gravedad de la inestabilidad muscular membrana y dañar 8. El uso de EBD para revelar daño de la membrana muscular es un parámetro de apoyo establecer similitudes del modelo animal para el estado de enfermedad humana 9. El poder de la EBD en el ratón ha llevado a varios laboratorios, incluido el nuestro, para desarrollar y aplicar EBD a los modelos de pez cebra de enfermedad neuromuscular. Debido a la aplicabilidad de análisis EBD, esta técnica se está aplicando activamente para corroborar modelos de pez cebra para el estado de enfermedad humana 11,15,22,24,32. Las larvas con las membranas musculares dañadas tendrá EBD absorción y fluorescencia roja, por lo tanto dentro de las fibras musculares. La fluorescencia observada en el espacio entre las fibras, pero no dentro de las fibras musculares individuales también pueden ser de carácter informativo de las fibras de desprendimiento de la membrana basal en tque la ausencia de daño de la membrana, proporcionando detalles de diagnóstico útil. Análisis EBD tiene aplicación potencial más allá de la validación de modelos animales. Los esfuerzos de nuestro laboratorio han demostrado recientemente que el análisis EBD es beneficioso en la validación de nuevos fármacos potencialmente terapéuticos 24. Determinar si los tratamientos terapéuticos potenciales reducir o abolir EBD captación en modelos de enfermedades neuromusculares puede significar acción terapéutica relevante 8. Este tipo de análisis puede ayudar a establecer el mecanismo (s) de la terapéutica y amplía la aplicación del análisis EBD. Al igual que con muchas técnicas, análisis EBD tiene varias advertencias que deben observarse durante el diseño y la práctica experimental. Por ejemplo, puede ser un reto para identificar el CCV debido a la engrosamiento del tejido con la edad. Además, es fácil dañar las larvas en la preparación antes y durante la inyección pericárdico, la reducción de los recuentos experimentales y el aumento de la necesidad de preparar un gran número de larvas.Además, el daño físico causado a las larvas durante la manipulación y la inyección podría dar lugar a falsos positivos como el músculo dañado puede tomar hasta EBD. Con el fin de superar algunos de estos obstáculos, hemos descrito una estrategia de co-inyección en este artículo de vídeo que permite la identificación fácil y fiable de las larvas con la infusión de colorante éxito inmediatamente después de la inyección y antes de posterior análisis. La controles co-inyección de FITC-dextrano para inyección éxito al permitir la confirmación de EBD en la vasculatura antes de su absorción por las fibras musculares. Esto puede ser particularmente útil como EBD fluorescencia se vuelve muy difusa en las larvas después de varias horas si no se recoge en las fibras musculares; como tal, puede ser difícil de detectar. Además, falta el CCV y la inyección de EBD en la yema o el cuerpo cavidad puede, después de la incubación, dar lugar a fluorescencia roja difusa similar a controlar embriones, sin embargo, con la reducción de la probabilidad de absorción por las fibras musculares dañadas. Colectivamente, estas cuevasats sugieren inyección EBD requiere paciencia y práctica para lograr resultados consistentes y fiables. En total, se describe un método práctico y sencillo para realizar el análisis EBD en larvas de pez cebra. Hasta la fecha, el uso del pez cebra como modelo de sistema, sobre todo como modelo de enfermedad humana, se ha expandido rápidamente. Esta expansión se debe en parte al desarrollo continuo y la modificación de las técnicas experimentales que mejoran sobre las ventajas actuales del sistema de pez cebra. La técnica de inyección EBD ofrece una herramienta adicional y potente al arsenal de un investigador para la validación y el estudio de modelos de enfermedad del músculo del pez cebra. La aplicación en curso y la modificación de esta técnica tiene el potencial de ayudar a descubrir nuevas estrategias terapéuticas, así como mecanismos de la enfermedad que causa.

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.

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	<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>
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		<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>
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		<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>
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</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.

La mezcla de inyección EBD se inyectó en el CCV de mutantes homocigotos sapje y hermanos de tipo salvaje en 3 dpf. Las inyecciones que llenaron las cámaras del corazón (Figura 1B) y luego se analizaron para la inyección con éxito mediante la visualización de FITC-dextrano en la vasculatura bajo fluorescencia verde (Figura 2). Después de un periodo de incubación de 4 horas, EBD captación fue examinado a nivel somito mediante microscopía de fluorescencia. Hermanos de tipo salvaje no mostraron fluorescencia EBD dentro de las fibras musculares visibles (Figura 3A), mientras que los mutantes homocigotos sapje mostraron EBD captación, lo que indica daños en la membrana muscular 15 (Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> Figura 1. mezcla de inyección Inyectar EBD en la vena cardinal común (CCV) de un embry pez cebrao. Embrión (A) no inyectados. Flecha indica la ubicación ideal para la inyección CCV. (B) la inyección exitosa en el CCV. El colorante entra en las cámaras del corazón (flecha) y comienza a ser bombeado a través de la vasculatura. (C) Una inyección CCV sin éxito dará lugar a algunos o todos el tinte de entrar en el saco vitelino del embrión (flecha). <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53183/53183fig2.jpg" /> Figura 2. Los embriones se pueden ordenar para inyección con éxito mediante la observación de la distribución-FITC dextrano bajo fluorescencia verde en toda la vasculatura inmediatamente después de la inyección y antes de la EBD captación.Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 3" src="/files/ftp_upload/53183/53183fig3.jpg" /> Figura 3: EBD será absorbido por las fibras con las membranas dañadas hermanos (A) Wildtype que no muestran fluorescencia EBD en las fibras musculares.. (B) sapje mutante homocigotos con fluorescencia EBD dentro de las fibras musculares múltiples (flechas). Todas las larvas fueron inyectados con la mezcla de inyección EBD y se analizó después de un período de incubación de 4 horas a 3 dpf. Hermanos y mutantes fueron ordenados por el músculo de fibra desprendimiento antes de la inyección CCV. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta 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.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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

Research

JoVE Journal

Developmental Biology

9.0K vistas.  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.

Video: 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.

La captura de Reparación de Tejidos en pez cebra larvas con Time-lapse Brightfield Estereomicroscopía

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 ...

Investigación

Biología del Desarrollo

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.

La inducción de la regeneración muscular esquelético aguda por inyección Cardiotoxin

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.

Dispositivos microestructurados para microinyección optimizado y la proyección de imagen de larvas de pez cebra

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.

Aplicación de la estimulación crónica para estudiar adaptaciones fenotípicas de músculo esquelético de rata inducida por actividad contráctil

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.

Identificación de células satélite del músculo esquelético por inmunofluorescencia con anticuerpos de laminina y 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.

Aislamiento, cultivo, caracterización y diferenciación de células progenitoras musculares humanas del procedimiento de biopsia muscular esquelética

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.

Realización de Xenoinjertos musculares esqueléticos humanos en ratones inmunodeficientes

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.

Aislamiento y diferenciación de los mioblastos primarios de los explantes musculares esqueléticos de ratón

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.

Inmunohistoquímica de montaje completo en embriones y larvas de pez cebra

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 la regeneración muscular en modelos de pez cebra de enfermedad muscular

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