This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

Declaración de Ética: NOTA: Todos los experimentos se llevaron a cabo de conformidad con las directrices del Hospital General de Massachusetts para el uso de animales en la investigación. 1. Preparación previa <ol><li> Determinar cuántos adultos de pez cebra 6-12 meses de edad para herir. Plan de lesionar 10-20% más pescado que se necesitará. Con el fin de minimizar la variabilidad, el uso edad igualada peces hermanos fueron criados en el mismo tanque de modo que son aproximadamente del mismo tamaño y tienen menos variabilidad genética. Hembras son mucho más fáciles de inyectar en comparación con el pez macho, debido a la mayor capacidad del abdomen. Sin embargo, los machos y las hembras dan resultados similares. </li><li> Al hacer el experimento por primera vez, trazar una curva dosis para determinar la dosis de gentamicina apropiado para una cepa específica de pescado. Pruebe cada nuevo lote de gentamicina antes de su uso en los experimentos ya que la pureza de gentamicina varía de lote a lote. Una dosificación adecuada debe determinarse mediante la evaluación expressiel de los marcadores de lesiones tales como lhx1a. (Ver Figura 1 A - D). </li><li> Prepare la solución de gentamicina. Solución madre de gentamicina puede ser almacenada en -20 ° C y se descongeló para su uso posterior. Por ejemplo, para un / kg dosis 80 mg (80 mg de gentamicina / kg de peso corporal) en un pez de pesaje 0,5 g, preparar 2 mg / ml de gentamicina en solución salina tamponada con fosfato como una solución de trabajo conveniente. Esto proporciona una 20 l de gentamicina para la inyección intraperitoneal en el pescado. NOTA: En general, 80 a 120 mg / kg de lograr buenos resultados, pero dosis tan bajas como 40 mg / kg puede ser apropiado. La gentamicina también se puede comprar en solución para minimizar la exposición peligrosa para el personal de laboratorio. NOTA: La gentamicina en dosis altas puede ser tóxico. Use guantes y máscara al pesar en polvo. </li><li> Preparar 100 ml de 0,016% de agua tricaína para anestesiar a los peces. Preparar un balance de 25x tricaína (4 g / 1 L) disuelto en agua estéril milliQ ajustar el pH a 7 y se almacena a 4 ° C hasta su uso. Diluir 4 ml en 100 ml de agua de pescado para una concentración de trabajo de 0,016%. NOTA: tricaína es un irritante de anestesia y la piel, use guantes al manipular. </li><li> Haz un pez cucharada de una pipeta de transferencia de plástico mediante la reducción de la bombilla en forma de cuchara y cortar 2 ranuras en la parte inferior para drenar el agua. Jeringas de 1 ml de carga (con 10 l gradaciones) con la solución de gentamicina preparado y adjuntar una aguja G 30½. Eliminar las burbujas de aire. Gire la aguja de la jeringa para asegurarse de que la punta en ángulo de la aguja está de espaldas y las marcas de jeringas están mirando hacia adelante y legible. Para las inyecciones de control, preparar una jeringa y aguja con PBS estéril. </li><li> Preparar una escala con una superficie limpia o pesar barco, así como toallas de papel para el secado de pescado y para la celebración de los peces para la inyección. </li><li> Preparar pequeños contenedores de medio litro con tapas individuales para la celebración de los peces para la inyección de observación post durante la noche. Las pequeñas jaulas de apareamiento de plástico transparentes son útiles para este propósito. Estascontenedores no deben ser blanco - idealmente deben ser claros para que los elencos epiteliales blancas derramadas por los peces se pueden observar en un banco de copa negro. Llene cada recipiente con agua suficiente pescado para los peces para nadar cómodamente. </li></ol> 2. La inyección intraperitoneal de Gentamicina <ol><li> Anestesiar a los peces en solución tricaína 0,016% en agua los peces. Espere a que la tasa de ventilación de enmalle para frenar y para que los peces ya no responder al tacto. </li><li> Recoja los peces usando la pala de pescado, pasando de la cabeza hacia la cola con el fin de evitar lesiones en las branquias o aletas. Coloque el pescado en toallas de papel para absorber el exceso de agua girando el pescado con cuidado en su lado y sacuda el exceso de agua de la primicia. </li><li> Recoja los peces de nuevo y lo coloca en el barco pesando en la escala cero y pesar el pescado. Redondea a la 0.25 g más cercano, y calcular la cantidad apropiada de gentamicina para inyectar. NOTA: Para un 0.5 g de pescado utiliza una inyección de 20 l al 80 mg / kg dosis. </li><li> Recoja los peces de nuevo y colóquela sobre una toalla de papel plegado seco. Si la inyección con la mano derecha, mantenga la toalla de papel en la mano izquierda y coloque la cabeza del pez señala a la izquierda, con el vientre de fácil acceso. </li><li> Mantenga la jeringa con la aguja en un ángulo de 45 ° a la piel del vientre, anterior a la cloaca. Empuje la aguja debajo de la piel, a continuación, disminuir el ángulo y deslice la aguja hacia adelante bajo la piel, evitando los órganos internos. Presione el émbolo de la cantidad adecuada, hacer una pausa para asegurarse de que ningún líquido está saliendo alrededor de la aguja, y luego retire la aguja. Si la celebración de los peces es un problema constante, apoyando los codos contra el torso para estabilizar ellos. NOTA: Use guantes al manipular la solución de gentamicina. </li><li> Caída de cada pez en un recipiente individual. Observar a los peces y asegurar su recuperación de la anestesia. Mantenga el pescado a 28,5 ° C durante la noche. NOTA: Si el pescado no revive inmediatamente, use un trans plásticofer pipeta para regar agua a través de sus branquias de revivirlo. </li><li> Usar la misma jeringa y la aguja cuando la inyección de pescado múltiple con la misma dosis de gentamicina. Deseche la jeringa y aguja usada en el recipiente previsto apropiado. </li></ol> 3. Publicar Observación La inyección de Lesiones <ol><li> Al día siguiente (después de la lesión 1 día), coloque el pescado se inyecta en su contenedor sobre una superficie oscura. Yesos blancos de tejido epitelial muerta excretado por los peces heridos deben ser visibles. (Ver Figura 1E - H). Si no hay moldes, ya sea el pescado no estaba lesionado por la inyección de gentamicina (ya sea la dosis era demasiado baja, o algunos de la gentamicina se filtró durante el proceso de inyección) o el pescado estaba gravemente herido que resulta en un bloqueo completo de los uréteres y de cloaca con el tejido desprendido. NOTA: Si el pescado no puede borrar los moldes de su cuerpo por lo general va a morir dentro de 2-3 días y por lo tanto inutilizable durante más tiempoensayos. Si no hay moldes son visibles, la eutanasia a los peces en el agua tricaína por inmersión durante al menos 10 minutos después detiene el movimiento de enmalle. </li><li> Si votas tejidos blancos son visibles, establezca el pescado a un lado y continuar revisando los otros peces. Una dosis adecuada de gentamicina resultará en el 80-90% de los peces de ser utilizable. Aunar los peces heridos y luego los divide en diferentes grupos de tratamiento si se desea. </li></ol> 4. Cuidado de pescado Recuperación <ol><li> Mantenga pez en el agua limpia y el medio ambiente con poca gente. El agua sucia y el hacinamiento dará lugar a infecciones y la muerte no intencional. Trate de mantener no más de 6 peces / 500 ml de agua peces y cambiar el agua diariamente si es posible. NOTA: Este es un volumen mínimo para la conservación de espacio o si el investigador desea para llevar a cabo experimentos utilizando los tratamientos con fármacos caros. </li><li> No alimentar a los peces hasta 3 días después de la lesión. Recuperación de peces no comen en un primer momento, y cualquier alimento en el tanque se descomponen y promover bacterias growth. A partir de 3 días después de la lesión, alimentar a una pequeña cantidad una vez al día. </li></ol> 5. Análisis de Lesionados Riñones <ol><li> Para la hibridación in situ para la expresión de ARNm lhx1a, los riñones se pueden cosechar a partir de peces en puntos de tiempo deseados. La eutanasia pez en el agua tricaína en hielo al menos 10 minutos hasta que el movimiento de enmalle se detiene, luego retire la cabeza con una hoja de afeitar y abrir la cavidad del cuerpo y eliminar los órganos internos con fórceps. </li><li> Deje el riñón en su lugar (órgano pigmentada unido a la pared dorsal del cuerpo) y fijar el pescado en 4% de paraformaldehído en PBS durante la noche. Diseccionar el riñón 15 y continuar con el estándar de hibridación in situ. <ol><li> Brevemente, lavar los riñones en PBST (tampón fosfato salino 0,5% de Tween 20), se permeabilizaron con proteinasa K en PBST (10 g / ml) durante 1 hora a temperatura ambiente. Post-fijar con paraformaldehído al 4%, y se lavó de nuevo con PBST. </li><li> Entonces pre-hibridar los riñones durante la noche a 68; ° C en tampón de hibridación (50% formamida, 5x SSC, 50 mg / ml de heparina, 500 mg / ml tRNA, 0,1% de Tween 20, pH 6,0). </li><li> Incubar las muestras con la sonda marcada con digoxigenina en tampón de hibridación, a continuación, lavar durante 5-10 min cada uno con 100%, 75%, 50%, 25% tampón de hibridación a 68 ° C, luego se trasladó a temperatura ambiente y lavar dos veces durante 30 min con 2x SSC con 0,1% de Tween 20, y lavar dos veces durante 30 min con 0,2 x SSC con 0,1% de Tween 20. </li><li> Equilibrar la muestra 3x 5 min en el MAB (0,1 M de ácido maleico, NaCl 0,15 M, pH 7,5) y se bloquearon durante la noche a 4 ° C en el MAB con 10% de suero de cabra. Entonces incubar las muestras durante la noche en anti-digoxigenina-AP Fab 1: 5000 en MAB. </li><li> Lavar el 5x muestra para 1 hr en MAB, y equilibrar 3x durante 15 min en NTMT (0,1 M Tris pH 9,5, 0,05 M MgCl 2, M NaCl 0,01, 50 l de Tween 20). </li><li> Luego tratar los riñones con NBT / BCIP para detectar la señal. Fijar los riñones con paraformaldehído al 4% y se incubaron en dimetilformamida a remove exceso de NBT / BCIP, blanqueada durante la noche en agua desionizada. Lavar los riñones y luego fotografiar en SAFT con un 50% de glicerol bajo un cubreobjetos. </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

El pez cebra es ideal para el estudio de la regeneración de órganos adultos incluyendo el adulto riñón mesonéfrico 6. Estudios recientes han aprovechado de marcadores moleculares y nuevas líneas reportero transgénicos para caracterizar mejor los pasos que se dan durante la regeneración de nefronas y lo que las células podrían ser responsables 7,12. Observación de desechos urinarios se ha utilizado durante más de un siglo para diagnosticar la enfermedad renal en humanos 16. Aq...

El riñón es esencial para la homeostasis de fluidos, la regulación de la presión arterial y la filtración de los residuos del cuerpo. Aunque los mamíferos son capaces de reparar nefronas existentes después de la lesión utilizando células epiteliales diferenciadas 1-4, que parecen carecer de una piscina de células madre reservadas 5 y son incapaces de formar nuevas nefronas de novo. En contraste a los mamíferos, peces adultos son capaces de formar nuevas nefronas largo de la vida adulta para apoyar el crecimiento de los peces y en respuesta a la lesión 6,7. El pez cebra, Danio rerio, es un organismo modelo de gran valor para el estudio de la regeneración de órganos 8-10 y tiene el potencial para proporcionar información de gran alcance en las aplicaciones de la ingeniería de la reparación de los riñones humanos. La Tg (Lhx1a: EGFP) transgén 11 se ha demostrado para etiquetar un conjunto de células progenitoras nefrona en el riñón adultos de pez cebra 12, sin embargo los mecanismos que controlan la respuesta de lhx1a + células a una lesión rEmain claro. La gentamicina es un antibiótico aminoglucósido ampliamente utilizado con efectos nefrotóxicos y ototóxicos conocidos en humanos 13. La inyección intraperitoneal de gentamicina es un método establecido de inducir lesión renal aguda en peces 6. Esta lesión en imita pescado la pérdida de epitelio tubular y la cicatrización de los glomérulos que se produce en los seres humanos después de la gentamicina sobredosis 14. La inducción de lesiones en el pez cebra por inyección de gentamicina es una manera conveniente de inducir una respuesta regeneración síncrono fuerte, con muchas nuevas nefronas producidos y de proceder simultáneamente a través de etapas de la formación, la proliferación y la diferenciación. Este protocolo detalla nuestro método de lesión robusta y reproducible en el pez cebra riñón adulto mediante la utilización de un proceso de selección visual no invasiva para reducir al mínimo los valores atípicos. Aprovechamos el hecho de que las lesiones con gentamicina conduce a la muerte del tejido renal epitelial y formatoión de cilindros tubulares renales, que luego se acumulan en masas en los conductos mesonéfricos y cloaca. Estos son pasados ​​por el pescado y se pueden observar visualmente en el agua. Esto nos permite a la pantalla para los peces con una fuerte lesión no letal, que luego pueden ser agrupados para su posterior experimentación. Reducir al mínimo el número de peces o no lesionado que mueren antes de llegar al punto final del experimento conduce a más uniforme y la recopilación de datos eficiente y análisis. Además, no se requieren dispositivos especiales o reactivos, por lo que este método rentable y adecuado para su uso en un entorno académico o la enseñanza. Usando este método, aquí mostramos las crecientes efectos de la dosis de gentamicina en la regeneración del riñón, así como el efecto del aumento de temperatura.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin sodium sulfate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">G1264</td>
			<td>TOXIC, purity varies from batch to batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic transfer pipets</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 ml Norm-Ject syringes</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">72520</td>
			<td>green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30G1/2 needles</td>
			<td style="width:101px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>IRRITANT</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">16% paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td>Make 4% in 1xPBS for working solution</td>
		</tr>
	</tbody>
</table>

kidney regeneration in adult zebrafish by gentamicin induced injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Lesión gentamicina puede ser confirmada visualmente por la observación de moldes epiteliales renales en el agua (Figura 1). Diferentes dosis de gentamicina se utilizaron para herir a adultos de pez cebra TUAB tipo salvaje, lo que resulta en un número creciente de lhx1a + agregados celulares en el riñón regeneración (Figura 1A - D). Yesos blancos pueden verse fácilmente en el agua 1 día después de la lesión (Figura 1E - H).</st...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

La regeneración renal en adultos de pez cebra por gentamicina lesión inducida

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Research

JoVE Journal

Developmental Biology

10.7K vistas.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 

Video: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Lesiones Embarcaciones mecánica en embriones de pez cebra

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Investigación

Biología del Desarrollo

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Ablación específico de hepatocitos en pez cebra para estudiar biliar impulsada regeneración hepática

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Desarrollo de un Modelo de hígado fibrótico inducida por etanol en el pez cebra para el Estudio de células progenitoras mediada por la regeneración de hepatocitos

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Cultura de adulto pez cebra transgénico de retina explantes de Células vivas imágenes de microscopía multifotónica

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Enfoque preciso de la ablación celular para modelar lesiones renales agudas en el desarrollo de pez cebra

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Análisis histológicos de lesiones hepáticas alcohólicas agudas en peces cebra

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Un método simplificado y eficiente para aislar Keratinocytes humanos primarios de tejido adulto de la piel

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Nanopartícula mediada por siRNA silenciamiento génico en corazón de pez cebra adulto

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Pez cebra adulto lesión modelos para estudiar los efectos de la prednisolona en regeneración de tejido óseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

Método de electroporación para la entrega in Vivo de ADN plásmido en el telencéfalo adulto de pez cebra

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...