Agradezco a Dr. Alan O. Perantoni (NIH/NCI/CDOBLE) para apoyar la presentación de este manuscrito. También agradezco el Dr. Michael Hall (NIH/NCI/CDOBLE) y Nirmala Sharma (NIH/NCI/CDOBLE) para la edición de este manuscrito. Agradezco a Lai Thang (SAIC) para su cría de ratón excelente. Este trabajo fue apoyado por los institutos nacionales de salud, Instituto Nacional del cáncer y centro de investigación del cáncer.

ratones (Wnt5a flox / flox ratones (Wnt5a tm1.1Tpy) y la línea Dll1Cre, modelo UVJO) 7 fueron manejados según pautas de NIH para el cuidado y uso de animales de laboratorio y estudió con un protocolo aprobado por el NCI Frederick Animal cuidado y uso.
 1. preparación de la solución de colorante azul de metileno <ol><li>medir 0,1 g de azul de metileno en polvo.</li>
	<li>Disolver el azul de metileno en solución salina normal 10 mL o 1 x de tampón fosfato salino (PBS) completamente por Vortex.</li>
	<li>Filtro con un filtro de jeringa (tamaño del poro de la membrana 0.45µm) para eliminar la obstrucción durante la inyección de la solución de azul de metileno 10 mg/mL.</li>
	<li>Montar un estéril cuero cabelludo vena set (27GX3/4 &#8243;) con una jeringa desechable de 3 mL y llene la jeringa con 3 mL filtrada azul de metileno solución. 
	Nota: Para evitar la presión hidrostática y el tinte posterior flujo, coloque la punta de la aguja sobre la jeringa llenada de solución de azul de metileno.</li>
</ol> 2. Disección de embriones prenatales <ol><li>limpio de disección, tijeras y pinzas con etanol al 70%.</li>
	<li>Para recolectar embriones perinatales en E18.5 o E19.5, eutanasia ratones embarazadas primero mediante inhalación de CO 2 y llevar a cabo la dislocación cervical según las pautas de NIH. 
	Nota: El ratón hembra embarazado generalmente da nacimiento partida en E18.5, sin embargo, los riñones más grandes son más fáciles de manipular. Por lo tanto, son más fáciles de realizar este análisis en riñones en E19.5 más cercano al nacimiento. Sin embargo, cachorros con troquel de obstrucciones urinarias bilateral después del nacimiento. Dependiendo de las características de los ratones experimentales, un día/hora de recogida apropiado debe determinarse empíricamente.</li>
	<li>Rociar etanol al 70% en la superficie ventral abdominal y luego abrir la cavidad abdominal ventral mediante disección tijeras y fórceps.</li>
	<li>Levantar el útero entero y separado del cuerpo cortando con tijeras en las puntas de los cuernos uterinos de disección.</li>
	<li>Lavar el útero entero con PBS 1 x en una placa Petri.</li>
	<li>Cortar el útero segmentally con tijeras de disección y retirar decidua placentaria con disección pinzas para exponer los embriones en sacos vitelinos.</li>
	<li>Primero retire el saco vitelino y después retire la membrana amniótica con fórceps para liberar los embriones perinatales de disección.</li>
	<li>Decapitar a un embrión con tijeras de disección. Limpie el exceso de sangre con gasas estériles. Precisar el embrión con su superficie ventral hacia arriba en un microscopio de disección equipado con una cámara digital para la proyección de imagen. Recoger la cola para genotipificación si es necesario. 
	Nota: Realizar un embrión las inyecciones a la vez.</li>
	<li>Abra cuidadosamente la cavidad abdominal del embrión con pinzas por el desgarramiento de la piel. Entonces, Remueva cuidadosamente el exceso órganos y tejidos como el hígado, el estómago y el intestino con fórceps por cortarlos o tirando de los mismos para exponer los riñones, los uréteres y la vejiga que se encuentran dorsalmente ( figura 1A). Absorber el exceso de sangre de embrión disecado con gasas estériles si fuera necesario. 
	Nota: Sangre exceso interfiere con la identificación de la pelvis renal para la inyección del tinte.</li>
</ol> 3. Inyección de colorante azul de metileno en la Pelvis Renal y el control de flujo de tinte <ol><li>quitar burbujas en la aguja y el tubo por expulsión de solución de azul de metileno de la punta de la aguja de presión hidrostática. Levantar la jeringa que contiene la solución de colorante por encima del nivel de la punta de la aguja para iniciar el flujo y luego baje la jeringa para detener flujo.</li>
	<li>Insertar la aguja en la pelvis renal cerca de uréter proximal, procurando no para lo una vez colocado molestar a. Realizar la inyección de colorante en un riñón para determinar la obstrucción del tracto urinario. 
	Nota: Ejecutar el tinte inyección en cualquier renal anormal 7 primera siguiendo la misma forma para determinar la obstrucción del tracto urinario.</li>
	<li>Levante la jeringa hasta unos 20 cm para proporcionar presión hidrostática y dejar 15 μl - 60 μL del flujo de la solución colorante. La tasa de flujo será aproximadamente 3 μl/s si la jeringa se eleva a esta altura el embrión.</li>
	<li>Monitorear el color azul del colorante a partir de primero en la pelvis renal, entonces en la longitud del uréter y finalmente en el lumen de la vejiga. 
	Nota: Tarda cerca de 5 s para ver un color de tinte débiles emergen dentro del lumen de la vejiga si el uréter se inserta correctamente en la vejiga. Permitir que el tinte que se acumulan en el sitio bloqueado cerca de 15 s si no hay color de tinte en el lumen de la vejiga aparece.</li>
	<li>Coloque la jeringa hasta la parada de tinte flujo y retire la aguja del riñón.</li>
	<li>Tomar imágenes de los riñones, el uréter y la vejiga trazado con solución colorante usando la cámara y programa la proyección de imagen conectan a un estereomicroscopio.</li>
	<li>Hacer un registro de hidronefrosis del riñón, hydroureter y la posición final de la solución de colorante en un cuaderno de laboratorio.</li>
	<li>Realizar la inyección del riñón contralateral como se describe en las secciones 3.1) a 3.5) y permitir el flujo del tinte cerca de 20 s en total. 
	Nota: La luz de la vejiga se llenará con solución colorante, indicado por un color azul fuerte, si el uréter se inserta adecuadamente en el lumen de la vejiga. Después de la evaluación de obstrucción de la Unión de las vías urinarias, los uréteres y la vejiga pueden ser fijados para seccionamiento.</li>
</ol>

<ol>
	<li>Rasouly, H. M., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+urinary+tract+development+and+disease.">Lower urinary tract development and disease.</a> Wiley Interdiscip Rev Syst Biol Med. 5, 307-342 (2013).</li><li>Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Box+Genes+in+the+Kidney+and+Urinary+Tract.">T-Box Genes in the Kidney and Urinary Tract.</a> Curr Top Dev Biol. 122, 245-278 (2017).</li><li>Bohnenpoll, T., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureter+growth+and+differentiation.">Ureter growth and differentiation.</a> Semin Cell Dev Biol. 36, 21-30 (2014).</li><li>Nicolaou, N., Renkema, K. Y., Bongers, E. M., Giles, R. H., Knoers, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic,+environmental,+and+epigenetic+factors+involved+in+CAKUT.">Genetic, environmental, and epigenetic factors involved in CAKUT.</a> Nat Rev Nephrol. 11, 720-731 (2015).</li><li>Tripathi, P., Wang, Y., Casey, A. M., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+canonical+Smad+signaling+in+ureteral+and+bladder+mesenchyme+causes+ureteropelvic+junction+obstruction.">Absence of canonical Smad signaling in ureteral and bladder mesenchyme causes ureteropelvic junction obstruction.</a> J Am Soc Nephrol. 23, 618-628 (2012).</li><li>Aoki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Id2+haploinsufficiency+in+mice+leads+to+congenital+hydronephrosis+resembling+that+in+humans.">Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans.</a> Genes Cells. 9, 1287-1296 (2004).</li><li>Yun, K., Perantoni, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydronephrosis+in+the+Wnt5a-ablated+kidney+is+caused+by+an+abnormal+ureter-bladder+connection.">Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection.</a> Differentiation. 94, 1-7 (2017).</li><li>Uetani, N., Bouchard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plumbing+in+the+embryo:+developmental+defects+of+the+urinary+tracts.">Plumbing in the embryo: developmental defects of the urinary tracts.</a> Clin Genet. 75, 307-317 (2009).</li><li>Murawski, I. J., Watt, C. L., Gupta, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+urinary+tract+defects+in+mice:+methods+to+detect+the+presence+of+vesicoureteric+reflux+and+urinary+tract+obstruction.">Assessing urinary tract defects in mice: methods to detect the presence of vesicoureteric reflux and urinary tract obstruction.</a> Methods Mol Biol. 886, 351-362 (2012).</li><li>Weiss, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephric+duct+insertion+requires+EphA4/EphA7+signaling+from+the+pericloacal+mesenchyme.">Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.</a> Development. 141, 3420-3430 (2014).</li></ol>

El autor no tiene nada que revelar.

Riñones de ratón son funcionales a partir de las E16.5 y una prueba de inyección de tinte es teóricamente posible a partir de este momento. Sin embargo, el riñón es demasiado pequeño para ser inyectada con la solución de tinte y fenotipos como hidronefrosis Hidrouréter no se observan claramente puesto que estos fenotipos son un efecto secundario de orina acumulado debido al transporte anormal de la orina. Estos fenotipos, de los obstáculos como UPJO o UVJO, son evidentes en E18.5 por distensión de los riñones...

El sistema urinario consiste en un par de riñones y uréteres comunes vejiga y uretra. La función principal del sistema urinario es mantener la homeostasis del cuerpo al administrar el equilibrio de agua y electrolitos de la sangre. Los riñones filtran la sangre para control las concentraciones de electrolitos y equilibrio acido-base y producen orina para excretar el exceso de agua y residuos incluidos los solutos y metabolitos. Orina es transportada a través del uréter de la pelvis renal del riñón a la bladderin una manera unidireccional donde se almacenan y finalmente eliminado a través de la uretra1.
Los uréteres son tubos rectos que se origina en el conducto nephric. Después de la brotación del conducto nephric en el día embrionario 10.5 (E10.5) en el ratón, el tallo del uréter se alarga y distingue a una estructura de varias capas llamada urotelio que es impermeable entre ratón E12.5 y E16.5. Las células mesenquimales que rodea el tallo del uréter también se diferencian en tres capas que consiste de internas células del estroma, células de grosor intermedio del músculo liso y fibroblastos adventitial externos. Se propagan las ondas peristálticas ureterales iniciando en la pelvis renal a través de la capa de músculo liso de la pared del uréter a la vejiga para el transporte de orina2,3, que se produce a partir de E16.5 en el ratón1.
Anomalías congénitas del riñón y vías urinarias (CAKUT) se encuentran entre las enfermedades genéticas más frecuentes, presentes en aproximadamente el 1% de fetos humanos1,4y compuesto de una variedad de fenotipos como hidronefrosis e Hidrouréter. La acumulación anormal de orina en el riñón y del uréter produce hydronephrotic del riñón e Hidrouréter formación. Una causa de hidronefrosis o Hidrouréter formación es una obstrucción de las vías urinarias. Obstrucción de la UUP (UPJO) es causada por el flujo de orina aberrantes debido a una obstrucción entre el uréter proximal y la pelvis renal, dando por resultado hydronephrosis y estrechamiento proximal Hidrouréter con forma angular o persistente plegable5, 6. Además, la inserción ectópica del uréter distal en la pared de la vejiga o el tracto reproductivo se denomina obstrucción ureterovesical (UVJO). UVJO también puede inducir la hidronefrosis y hydromegaureter de7,de formación8. Un defecto de unión (UVJ) ureterovesical adicional es el reflujo vesicoureteral (RVU). VUR se caracteriza por flujo retrógrado de orina desde la vejiga hacia el riñón en UVJ. En comparación con UVJO, perinatales embriones con RVU no claramente muestran un riñón hydronephrotic dilatado o Hidrouréter severa fenotipo9.
En el ratón de laboratorio, flujo de orina puede ser examinado por la inyección de un colorante, como azul de metileno, en la pelvis renal9. La solución de azul de injectedmethylene traza la trayectoria de la orina desde la pelvis renal a través del uréter y la vejiga. La hidronefrosis puede ser reconocida por una expansión del tinte en el riñón. UPJO puede ser detectado como una obstrucción del flujo del tinte en el uréter proximal con pelvis renal dilatada5. Cualquier dilatación del uréter indicada un diámetro ensanchado muestra un ejemplo de Hidrouréter. Finalmente, acumulación de tinte en la pared de la vejiga o en el sitio del tracto reproductivo indica UVJO con riñón hydronephrotic distendido y dilatado hydromegaureter7,10. Para detectar el RVU, la solución de tinte se inyecta en la vejiga y el posterior flujo retrógrado es monitoreado en el riñón9.
Aquí, se presenta un protocolo para la inyección de tinte de azul de metileno en la pelvis renal de un embrión perinatal. Este protocolo permite el rastreo de flujo de la orina desde la pelvis renal a través del uréter y la vejiga y verifica posibles obstrucciones de cruce vías urinarias tales como UPJO o UVJO.

<table width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="width:295px;height:21px;">Methylene blue</td>
			<td style="width:197px;">Sigma-Aldrich</td>
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assessing urinary tract junction obstruction defects by methylene blue dye injection

Defectos de la obstrucción urinaria cruce son anomalías congénitas inducción de hidronefrosis e Hidrouréter. Defectos de obstrucción urinaria murino cruce pueden evaluarse mediante el seguimiento flujo de tinte de azul de metileno dentro del sistema urinario. Se inyecta azul de metileno en la pelvis renal de riñones embrionarios perinatales y flujo del tinte es monitoreado desde la pelvis renal del riñón por el uréter y en el lumen de la vejiga después de aplicar presión hidrostática. Acumulación de tinte será evidente en el lumen de la vejiga del tracto urinario perinatal normal, pero será limitada entre la pelvis renal y el punto final de un uréter anormal, si se producen obstrucciones del tracto urinario. Este método facilita la confirmación de obstrucción de vías urinarias cruce y visualización del hydronephrosis y del hydroureter. Este manuscrito describe un protocolo para la inyección de tinte de azul de metileno en la pelvis renal para confirmar obstrucciones de cruce de las vías urinarias.

Los riñones y sistema urinario inferior se encuentran dorsal a la mayoría de los otros órganos internos como el hígado y el intestino. Después de quitar estos otros órganos internos, un par de riñones y los uréteres y la vejiga de una sola son visibles (figura 1A). Sobre disección exitosa, fluirá el colorante inyectado en la pelvis renal del riñón en la vejiga vía el uréter. La pelvis renal es la forma de embudo parte proximal dilatada del urét...

Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Inyección de colorante azul de metileno en la pelvis renal facilita la evaluación de los defectos de obstrucción urinaria cruce durante el desarrollo embrionario las vías urinarias del ratón. Aquí, se describe un protocolo para la inyección de colorante azul de metileno en la pelvis renal.

Evaluación defectos de obstrucción urinaria cruce por la inyección de tinte de azul de metileno

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

assessing-urinary-tract-junction-obstruction-defects-methylene-blue

Research

JoVE Journal

Biology

15.5K vistas.  National Institutes of Health. Inyección de colorante azul de metileno en la pelvis renal facilita la evaluación de los defectos de obstrucción urinaria cruce durante el desarrollo embrionario las vías urinarias del ratón. Aquí, se describe un protocolo para la inyección de colorante azul de metileno en la pelvis renal.

Video: Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Disección y de grabación de la unión neuromuscular C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Investigación

Biología

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Cerebro del pez cebra ventrículo inyección

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Implantación de Ferumoxides Etiquetada células madre mesenquimales humanas en los defectos del cartílago

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi interferencia por inyección de dsRNA en Drosophila Los embriones

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Un modelo de ratón de la obstrucción Intestinal parcial

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Un ensayo de comunicación Unión intercelular yoduro amarillo fluorescente proteína-Gap

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Método Ex Vivo para evaluar la motilidad espontánea del tracto reproductivo de ratón y un algoritmo de seguimiento de movimiento del útero basado en MATLAB para el análisis de datos

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...