Agradecemos a los miembros del laboratorio de Wingert útil para los debates de la biología y las técnicas de AKI pez cebra, y Diep C. por compartir su receta de metilcelulosa. Los autores también desean expresar nuestra gratitud a los miembros del personal del Centro de Notre Dame para la Investigación de pez cebra para proporcionar una excelente atención permanente para la cría de nuestra colonia de pez cebra. La financiación del premio NIH NIDDK conceder DK083512 y de laboratorio generosa financiación inicial de la Universidad de Notre Dame, apoya este trabajo.

1. Microinjection procedure to label the zebrafish pronephros proximal tubule epithelium
For this procedure, we use a microinjection system (Harvard Appartus, PLI90), with pressurized air supplied by an air compressor (Jun-Air, Model 6-4), and micromanipulator apparatus (Narishege parts MN151, IP3, and GJI) that are assembled at a stereomicroscope station (Nikon, SMZ-Zoom 645) as per manufacturer instructions. Dextran conjugates are readily endocytosed by the nephron proximal tubule, enabling preferential labeling of this epithelial cell population9.
<ol>
 <li>Exposure to methylene blue can accentuate the autofluorescence of the yolk sac and makes visualization of the nephron tubules more difficult.</li>
 <li>Raise the fertilized embryos at 28.5&deg;C to a developmental timepoint between 48-55 hours post fertilization (hpf). This is the ideal stage to perform larvae microinjections because of the ease at which intramuscular microinjection can be performed, and because renal function commences at this time.</li> 
 <li>Remove the chorion from any unhatched zebrafish using a pair of fine forceps. Rinse the chorion debris from the petri dish and refill the dish with 30 mLs of modified E3 solution.</li>
 <li>Prepare an injection tray for the embryos. Our preferred injection mold is made of 1.5% agarose/E3, in which triangular depressions are formed such that the embryo can be positioned inside for injection. To make the injection mold, we float a plastic mold with wedge shaped protrusions (described in detail previously11) in a Petri dish filled with a base of 1.5% agarose.</li>
 <li>Prepare glass microinjection needles by pulling capillary tubes with a needle puller, as described in the Zebrafish Book10. In brief, pull the glass capillary tubes then use fine metal forceps to cut the tip of the microinjection needle to make a tip with a sharp point while viewing the needle tip under a stereomicroscope. Store the cut needles covered in a petri dish and positioned on modeling clay to protect the cut edge and prevent dust accumulation.</li>
 <li>Prepare the injection solution to label the proximal tubule. Dissolve 40 kD dextran-fluorescein conjugate (Invitrogen, D1845) at a concentration of 1 mg/mL in distilled water.</li> 
 <li>To anesthetize the fish, add 5 mLs of 0.2% Tricaine pH 7.0 to the petri dish containing the zebrafish larvae in 30 mLs of E3. The fish are anesthetized when they no longer exhibit the touch response. It is vital that the fish are completely anesthetized or they will twitch upon attempting to inject them.</li>
 <li>Transfer the anesthetized fish to an injection mold tray using a plastic transfer pipet. Position the animal with its head in the deepest part of the triangular depression well, and on its side such that the trunk is resting along the angled side of the well.</li> 
 <li>Perform intramuscular microinjection. Load a cut needle with 2-3 &mu;L of dextran fluorescein, and inject the animal in a trunk somite with approximately 1 nL of solution. Aim for the upper portion of the somite, and avoid the yolk sac extension. This ensures that the nephron tubules are not mechanically disrupted by the microinjection process.</li>
 <li>Transfer injected zebrafish to a clean petri dish, and add modified E3 solution. Rinse the animals 3 times with fresh E3 to remove all traces of Tricaine. Cover the lid of the petri dish with aluminum foil to provide light-protection for the dextran, and return the animals to the 28&deg;C incubator overnight.</li>
</ol>
2. Targeted laser ablation of proximal tubule cells
For this procedure, we use a stereomicroscope with epifluorescence to score animals with dextran fluorescein brightly-labeled proximal tubules and select these for laser ablation. Then, we use a pulsed micropoint laser system (Photonic Instruments, Inc) that has been attached to a compound microscope (Nikon Eclipse 80i with epifluorescent attachment), and calibrated for use as per manufacturer instructions, to perform nephron cell ablation. We have had the most success in cell ablation by using a FITC filter that enables the researcher to view the fluorescent tubule regions under brightfield lighting.
In advance of this procedure, prepare immobilization media for the ablation by dissolving 1.5% methylcellulose in 0.02% tricaine/E3. Store aliquots of methylcellulose for immediate use at room temperature, or at 4&deg;C for long-term storage.
<ol>
 <li>Anesthetize the 72 hpf zebrafish by adding 5 mLs of 0.2% Tricaine pH 7.0 to the petri dish containing the zebrafish larvae in 30 mLs of E3. The fish are anesthetized when they no longer exhibit the touch response. It is vital that the fish are completely anesthetized or they will twitch upon attempting to perform the laser ablation.</li>
 <li>Examine the injected animals under the appropriate fluorescent setting and select animals in which you can easily visualize the proximal tubules.</li>
 <li>Transfer selected embryos to a separate dish containing tricaine. Animals can be anesthetized for up to 30 minutes before performing cell ablation. If you score &gt;15 animals, return these to a separate dish containing fresh modified E3 while awaiting ablation as each cell ablation takes several minutes.</li>
 <li>To perform the ablation, transfer one anesthetized zebrafish to a small petri dish containing 1.5% methylcellulose/0.02% tricaine for 2 minutes to wash the embryo in immobilization media.</li> 
 <li>Next, transfer the embryo to a glass depression slide containing a drop of 1.5% methylcellulose/0.02% tricaine. Gently position the animal with a fine probe such that its ventral side faces the slide and dorsal side is facing up.</li> 
 <li>Place the slide on the compund microscope stage, and focus on the dorsal side of the animal. Perform ablation of the nephron cells under focus by depressing the foot pedal. You will see dispersal of the fluorescence as the renal epithelial cells are ablated (Figure 1A).</li> 
 <li>Gently transfer the ablated animal to a well in a 12 well-dish for subsequent culturing. Rinse the E3 2-3 times to wash away the methylcellulose.</li>
 <li>After injection, raise the animal to the desired timepoint and perform desired analysis as per established histological and molecular techniques for young zebrafish embryos (Figure 2).</li>
</ol>
3. Representative results: 
The data that are shown in Figure 1 indicate the ablation timecourse. After intramuscular injection with dextran-conjugates, the proximal tubule is preferentially labeled. Injection of dextran-FITC was used to label the kidney for laser ablation, and ablated animals were reinjected with dextran-rhodamine at one additional timepoint following ablation to reimage the proximal tubule population. Expression analysis techniques like in situ hybridization can be used to analyze changes in fixed samples at single timepoints following cell ablation, as shown in Figure 2. 
<img src="/files/ftp_upload/2845/2845fig1.jpg" alt="Figure 1"/> 
Figure 1: Laser ablation procedure is followed by rapid regeneration of the tubule epithelium. (A-C) Timecourse of cellular changes during and subsequent to laser ablation of zebrafish larva proximal tubules at time of ablation (day 3, panel A), or one or three days post ablation (day 4, panel B; day 7, panel C). (Top) schematics show position of the nephron, with dextran-FITC (green) and dextran-rhodamine (red), and (bottom) live images of control and laser-ablated (LA) nephrons. 
<img src="/files/ftp_upload/2845/2845fig2.jpg" alt="Figure 2"/> 
Figure 2: Analysis of gene expression following laser ablation. Following laser ablation at day 3, embryos were fixed and processed by whole mount in situ hybridization to detect transcripts for slc20a1a, which mark the proximal convoluted tubule segment of the nephron. (A) A control embryo that was not subjected to laser ablation, (B) an embryo in which a large stretch of tubule epithelium was ablated, and (C) an embryo in which a short interval of tubule epithelium was ablated. Black line indicates the extent of the proximal tubule in A to provide reference, blue lines indicate the extent of laser ablation in panels B-C, and * indicates the control (non-ablated) contralateral nephron in panels B-C.

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No hay conflictos de interés declarado.

El pez cebra es un organismo modelo ampliamente utilizado en muchos aspectos de la ciencia, las aplicaciones de las que están creciendo 6. El empleo del sistema de modelo de pez cebra en el estudio de enfermedades renales como la IRA ha llevado a importantes observaciones y seguirá siendo un buen modelo para estudiar la lesión renal 7,8. Con un genoma que se ha secuenciado y muchos protocolos moleculares en su lugar, se ha convertido cada vez más fácil para llevar a cabo la investigación que utiliza pez cebra sofisticados modelos transgénicos, derribo y estudios de genética misexpression. El pez cebra tiene un ciclo vital corto con un tamaño de la generación de grandes y bien definidas las etapas de desarrollo. Además, las etapas de embriones y larvas de pez cebra son en gran medida transparente, por lo que los estudios de imagen posible sin la disección del organismo. A lo largo de las etapas embrionarias y larvas, el pez cebra tiene un riñón pronephric compone de dos nefronas, que siguen siendo visibles en estos puntos de tiempo de desarrollo. Pez cebra nefronas pronephric son análogos en estructura y función a sus homólogos de los mamíferos, por lo que es un buen modelo para estudios de toxicidad aguda insuficiencia renal 4,5. El riñón es crucial para la supervivencia de los seres humanos y otros vertebrados, que actúa como el órgano que limpia el cuerpo de los desechos metabólicos líquido. Esta función de limpieza se basa en la capacidad de las nefronas del riñón para filtrar la sangre, y luego modificar el filtrado para recoger los desechos nitrogenados en la secreción y al mismo tiempo mantener el equilibrio de líquidos y electrolitos. Nefronas se componen de un filtro de la sangre, los túbulos epiteliales, y la fuga en un conducto. La integridad estructural y funcional de las tres partes es esencial para la función de nefronas. Diversos insultos a los riñones, incluyendo la exposición a nefrotoxinas, la isquemia, o la obstrucción del tracto de salida urinaria puede dar lugar a un IRA. La patología de la lesión renal aguda se asocia a menudo con 1,2 necrosis tubular aguda. Los estudios clínicos han demostrado que el consiguiente insuficiencia renal tiene una mortalidad que puede oscilar entre 7-80% dependiendo de la causa y el contexto de la insuficiencia renal. Sin embargo, el diagnóstico rápido y la inversión de la causa subyacente de la IRA es cada vez más asociado con la recuperación a través de la regeneración de los túbulos nefrona necrótico. Recientes estudios de mapeo destino han demostrado que la población de células grandes que vuelve a llenar dañado túbulos nefrona son las células epiteliales que se originan en las áreas adyacentes, sin lesiones 12. Estos hallazgos no han eliminado la posibilidad de que renal células madre / progenitoras pueden participar en el proceso, y los informes independientes han sugerido que las células derivadas de médula ósea pueden contribuir a la regeneración renal 13. Está claro que continuaban las investigaciones son necesarias para resolver mejor las fuentes celulares de la regeneración del epitelio renal. Tanto la regeneración y el desarrollo de nefronas comparten el fenómeno de los interruptores en el estado entre los fenotipos celulares mesenquimales y epiteliales. Durante el desarrollo de nefronas, las células mesenquimales precursoras se diferencian en un fenotipo fijas, correspondientes a una membrana basal para formar el epitelio tubular 3. El fenómeno migratorio especulado de las células epiteliales después de la insuficiencia renal aguda requiere una desdiferenciación en un fenotipo mesenquimal. Curiosamente, los estudios recientes sugieren que las células mesenquimales en los riñones dañados presentan un perfil de expresión similar a la de las células mesenquimales en el desarrollo 14. Varios informes han detectado cambios en las moléculas de adhesión celular, proteínas de la matriz, citocinas y quimiocinas. A raíz de la propuesta para la transición mesenquimal epitelial, las células montamos sobre una membrana basal tubular y reiniciar las actividades del epitelio. La determinación de los genes importantes en este proceso sigue siendo un tema de investigación de nuestro laboratorio y otros. Gran parte del trabajo para dilucidar los factores clave en este proceso que queda por hacer, pero los avances significativos en el campo ha sido anunciada por el estudio de los efectos de la gentamicina. AKI y fallo renal causado por la gentamicina es relevante teniendo en cuenta la prevalencia de los compuestos nefrotóxicos utilizados en la medicina 15. Se utiliza como un tratamiento para gram-negativos infecciones bacterianas, la administración de aminoglucósidos conduce a una lesión aguda en el 10-25% de los casos. La droga causa una gran cantidad de efectos negativos sobre el celular, así que resulta en la apoptosis o necrosis en muchas células tubulares. Los estudios han encontrado que la droga se unen preferentemente a un complejo formado por megalina y cubulin que está involucrado en la endocitosis. Estas moléculas se encuentran en abundancia en las células epiteliales del túbulo proximal, aunque no se limitan a la expresión en estas células. A través de la señalización celular que resulta en la inducción de estrés oxidativo, la liberación de vasoconstrictores, el agotamiento de ATP celular, la hipoxia, la inhibición de las fosfolipasas, los antibióticos gentamicina y similares causanapoptosis y necrosis en las células epiteliales. Además, los antibióticos desencadenar la contracción mesangial en la sangre nefrona filtro (glomérulos), lo que resulta en una caída en la tasa de filtración glomerular y negativamente alterando la capacidad del riñón para filtrar la sangre. La producción de especies reactivas del oxígeno, moléculas inmunoestimulantes, y la activación de las fosfolipasas tienen efectos difusos en la nefrona, la creación de una catastrófica patología que lleva a la insuficiencia renal aguda. Mientras que los estudios con antibióticos gentamicina y similares han sido y seguirán siendo herramientas importantes para la investigación renal regeneración, carecen de una precisión que puede ser útil en el tratamiento de determinadas cuestiones. Nuestro sistema de usar el pez cebra, junto con un método de ablación láser se describe en este protocolo responde a la necesidad de una herramienta de investigación precisa. La ablación con láser permite a los investigadores para inducir la destrucción de las células en las áreas focales del túbulo renal, que van desde una pequeña (2-3) a grandes (&gt; 50 a 100) la población, con una precisión inigualable. En este protocolo, utilizamos un método previamente desarrollado de la exposición a dextrano conjugados para etiquetar preferentemente poblaciones túbulo proximal del epitelio. Por otra parte, el pez cebra transgénico líneas que expresan la proteína verde fluorescente en una o más regiones del túbulo se podrían utilizar. Por ejemplo, cadherin17: eGFP pez cebra transgénico presentan fluorescencia fuerte en las regiones del túbulo distal (aunque débil en las poblaciones proximal), y podría ser valiosa para los estudios de regeneración en los segmentos de los túbulos otros 16. La visibilidad de la nefrona permitirá lapso de tiempo de grabación de vídeo de los cambios celulares en el tiempo. Cuando se combina con estudios de expresión génica, tales como montar toda la hibridación in situ o inmunohistoquímica, los investigadores pueden detectar alteraciones moleculares en la nefrona en el tiempo. En conjunto, estas estrategias se pueden empezar a formular una comprensión más dinámica de los acontecimientos que suceden en las células epiteliales renales son destruidos. La principal advertencia de nuestro modelo de ablación con láser es que puede recapitular aspectos parciales de las condiciones fisiológicas que suceden en humanos AKI. La muerte celular inducida por los daños físicos instantánea es diferente de la cascada de eventos que conduce a la necrosis de las células, y como tal, los factores humorales en estos microambientes diferentes no pueden ser idénticos. Además, existe evidencia de la apoptosis inducida durante el daño renal, y no se sabe si tales consecuencias transpirar insulto siguientes con un láser. Sin embargo, al igual que el cultivo celular es una herramienta que mejor responde a algunas preguntas que requieren un entorno muy controlado, la ablación por láser servirá como un muy controlado en el modelo in vivo de la IRA. Por lo tanto, las percepciones recogidas de estudios de la ablación renal epitelial en el pez cebra es probable que promueven el descubrimiento de las ideas fundamentales que se pueden aplicar a las investigaciones de otras lesiones renales y, potencialmente, para crear un mejor diagnóstico en seres humanos.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Platos de embriones </td><td> Halcón </td><td> 35-1005 </td></tr><tr><td> Pinzas de disección </td><td> Roboz </td><td> RS-5010 </td></tr><tr><td> Agujas de inyección </td><td> Sutter Instrumentos </td><td> BF100-50-10 </td></tr><tr><td> Agarosa ultrapura </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> (40 kDa) Dextran-fluoresceína </td><td> Invitrogen </td><td> D1845 </td></tr><tr><td> Pipeta de plástico </td><td> Samco Científico, Fisher </td><td> 204, 13-711-23 </td></tr><tr><td> Tricaína </td><td> Sigma </td><td> E10521 </td></tr><tr><td> Metilcelulosa </td><td> Sigma </td><td> M0262 </td></tr><tr><td> La depresión de diapositivas </td><td> Pescador </td><td> S175201 </td></tr><tr><td> De 12 y plato </td><td> Corning </td><td> 3512 </td></tr></tbody></table>

laser ablation of the zebrafish pronephros to study renal epithelial regeneration

La lesión renal aguda (IRA) se caracteriza por altas tasas de mortalidad de deterioro de la función renal durante un período de horas o días que culmina en insuficiencia renal 1. AKI puede ser causada por una serie de factores, incluyendo la isquemia, las drogas basadas en la toxicidad o lesiones obstructivas 1. Esto se traduce en una incapacidad para mantener la homeostasis de fluidos y electrolitos. Mientras AKI se ha observado durante décadas, a partir terapias clínicas aún no se han desarrollado. Curiosamente, algunos pacientes con lesión renal aguda recuperar la función renal con el tiempo, un fenómeno misterioso que sólo se ha caracterizado rudimentally 1,2. La investigación que utiliza modelos de mamíferos de la IRA ha demostrado que los riñones isquémicos o nefrotoxina heridos-experiencia de la muerte de las células epiteliales de los túbulos nefrona 1,2, las unidades funcionales del riñón que se componen de una serie de áreas especializadas (segmentos) de tres tipos de células epiteliales . Dentro de las nefronas, la muerte de las células epiteliales es mayor en las células del túbulo proximal. Hay evidencia que sugiere que la destrucción celular es seguido por desdiferenciación, proliferación y migración de las células epiteliales circundantes, que pueden regenerar completamente la nefrona 1,2. Sin embargo, hay muchas preguntas sin respuesta acerca de los mecanismos de la regeneración del epitelio renal, que van desde las señales que modulan estos eventos a los motivos de la amplia variedad de habilidades entre los seres humanos para regenerar riñones lesionados. La larvas de pez cebra ofrece un excelente modelo para estudiar la regeneración del epitelio renal como su riñón pronephric se compone de nefronas que se conservan en los vertebrados superiores, incluyendo mamíferos 4,5. Las nefronas de las larvas de pez cebra se pueden visualizar con técnicas de fluorescencia debido a la relativa transparencia de los jóvenes pez cebra 6. Esto proporciona una oportunidad única de celda de la imagen y los cambios moleculares en tiempo real, en contraste con los modelos de mamíferos, donde nefronas son inaccesibles porque los riñones son sistemas estructuralmente complejos interiorizarse dentro del animal. Estudios recientes han utilizado la gentamicina aminoglucósidos como un agente tóxico causante de estudio de la lesión renal aguda e insuficiencia renal posterior: la gentamicina y otros antibióticos se ha demostrado que causan IRA en los seres humanos, y los investigadores han formulado métodos para utilizar este agente para provocar daño a los riñones en el pez cebra 7 , 8. Sin embargo, los efectos de la toxicidad de los aminoglucósidos en las larvas de pez cebra son catastróficos y mortales, que presenta una dificultad en el estudio de la regeneración epitelial y función en el tiempo. Nuestro método se presenta el uso de la ablación de células específicas como una nueva herramienta para el estudio de la lesión epitelial en el pez cebra. La ablación con láser ofrece a los investigadores la capacidad de inducir la muerte celular en una población limitada de las células. Otros campos de las células pueden ser escogidos en base a la ubicación morfológica, la función, o incluso la expresión de un fenotipo celular particular. Por lo tanto, la ablación por láser aumentar la especificidad de lo que los investigadores pueden estudiar, y puede ser un enfoque nuevo y poderoso para arrojar luz sobre los mecanismos de regeneración del epitelio renal. Este protocolo se puede aplicar ampliamente a llegar a las poblaciones de células en otros órganos en el embrión de pez cebra para el estudio y la regeneración de lesiones en cualquier número de contextos de interés.

Watch this Scientific Journal Video about Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration at JoVE.com

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

La lesión renal aguda (IRA) en los seres humanos es un problema clínico común causada por el daño a las células epiteliales que forman nefronas del riñón, y la lesión renal aguda se asocia con altas tasas de mortalidad del 50-70% 1. Tras la destrucción de las células epiteliales, las nefronas tienen una capacidad limitada de regeneración, aunque los mecanismos y las limitaciones que rigen este fenómeno se conoce bien. En este artículo de vídeo, describimos nuestra técnica de ablación láser dirigida de las células de la nefrona renal en el riñón del embrión de pez cebra, o pronefros. Nuestro nuevo método puede ser utilizado para complementar la nefrotoxicidad inducida por los modelos de IRA y obtener un conocimiento de alta resolución de la célula y las alteraciones moleculares que están asociados con la regeneración del epitelio de la nefrona renal.

La ablación con láser de la pronefros pez cebra para el Estudio de la regeneración epitelial renal

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

laser-ablation-zebrafish-pronephros-to-study-renal-epithelial

Research

JoVE Journal

Biology

15.2K vistas.  University of Notre Dame. La lesión renal aguda (IRA) en los seres humanos es un problema clínico común causada por el daño a las células epiteliales que forman nefronas del riñón, y la lesión renal aguda se asocia con altas tasas de mortalidad del 50-70% 1. Tras la destrucción de las células epiteliales, las nefronas tienen una capacidad limitada de regeneración, aunque los mecanismos y las limitaciones que rigen este fenómeno se conoce bien. En este artículo de vídeo, describimos n...

Video: Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

La neurogénesis cortical: La transición de los avances en el Laboratorio de terapias basadas en células

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Los estímulos generados por ordenador Modelo animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Procedimiento murino Trasplante Renal

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Métodos para el Estudio de la barbo pez cebra maxilar

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Usando la respuesta de optocinético para estudiar la función visual del pez cebra

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema para el cultivo de células epiteliales del pigmento del iris para el Estudio de Regeneración del objetivo de Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

La aplicación de ácido retinoico para obtener cultivos de osteocitos principal del ratón osteoblastos

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...