Name: dissection and observation of honey bee dorsal vessel for studies of cardiac function
Uploaded: 2016-12-12T13:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

The authors thank Drs. Jeffrey Bloomquist and Daniel Swale for their technical comments and suggestions. This project was partially funded by the Department of Entomology and the College of Agriculture and Life Sciences at Virginia Tech.

1. Recogida y preparación de los sujetos de prueba <ol><li> Recoger el número apropiado de las abejas de la colonia. NOTA: El número necesario depende no sólo del tamaño y alcance del experimento, sino también la habilidad del investigador. Por ejemplo, si hay 2 grupos de tratamiento con un tamaño deseado de la muestra de 10 abejas por grupo, un investigador experto razonablemente podría recoger un mínimo de 30 abejas para dar cuenta de las disecciones sin éxito y terminar con 20 videos útiles para marcar. </li><li> Minimizar la cantidad de tiempo que transcurre entre la recolección y la disección. NOTA: A pesar de abejas pueden ser alojados en el laboratorio para día antes de la disección, la tasa de éxito de las disecciones (es decir, la probabilidad de que el mantenimiento de un ritmo cardíaco estable en un vaso dorsal diseccionado) se ha observado para disminuir con relación a la cantidad de tiempo que las abejas se encuentran fuera de la colonia. <ol><li> Proporcionar abejas con una fuente de agua y alimentos, mientras alojados en el laboratorio. para example, como mínimo, proporcionar acceso a un 50% de solución de sacarosa en agua (w / v) (esto es suficiente para duraciones de menos de 6 h). Para períodos más largos, proporcionan acceso a las abejas de miel. </li><li> abejas de la casa de la noche a la mañana de laboratorio a una temperatura de aproximadamente 32 ° C y 60-80% de humedad relativa para reducir el estrés y evitar la deshidratación. </li></ol></li><li> Antes de la disección, anestesiar abejas brevemente para ayudar en la manipulación. NOTA: Esto puede disminuir la tasa de éxito de las disecciones y reducir el rendimiento. <ol><li> Enfriar las abejas ya sea colocándolos en hielo o en un refrigerador durante el tiempo suficiente para reducir el movimiento con el fin de ayudar en el manejo. </li><li> Alternativamente, exponer brevemente las abejas a CO 2 con el fin de ayudar en el manejo. NOTA: La exposición prolongada al frío puede reducir la tasa de éxito de las disecciones. Extendido o repetida exposición al CO 2 también puede reducir la tasa de éxito de las disecciones. </li></ol></li></ol> 2. DisecciónDorsal de la pared abdominal NOTA: Las abejas deben estar vivos en el momento de la disección. <ol><li> El uso de pinzas y / o tijeras de microdisección, eliminar patas y las alas para facilitar la disección del abdomen. Mantenga un pequeño vaso de precipitados o recipiente similar lleno de agua destilada cercano con el fin de enjuagar los instrumentos entre disecciones. </li><li> Mientras que restringir la abeja con fórceps, utilizar las tijeras de microdisección para cortar lateralmente a lo largo de la pared abdominal dorsal entre la primera y segunda tergites (véase la Figura 1). </li></ol><img alt="Figura 1" src="/files/ftp_upload/55029/55029fig1.jpg" /> Figura 1: Vista dorsal del abdomen de la abeja. La incisión inicial debe hacerse entre la primera y segunda tergites, como se indica mediante la línea roja. Barra de escala = 1 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig1large.jpg" target="_blank">Por favor, haga clic en ella</a>e para ver una versión más grande de esta figura. <ol start="3"><li> Aunque ligeramente agarrando el borde posterior de la segunda Tergito con las pinzas, corta longitudinalmente a lo largo de cada lado de la abeja de la incisión inicial a la aguijón (ver Figura 2). Tenga cuidado al cortar para evitar perforar el tubo digestivo. </li></ol><img alt="Figura 2" src="/files/ftp_upload/55029/55029fig2.jpg" /> Figura 2: Vista lateral del abdomen de la abeja. El segundo y tercer incisiones deben hacerse a lo largo de cada lado del abdomen, como se indica mediante la línea roja. Barra de escala = 1 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <ol start="4"><li> Intercambiar las tijeras por un segundo juego de pinzas finas y utilizarlos para separar cuidadosamente la pared abdominal dorsal del resto of abdomen. Retire con cuidado el aguijón y cualquier parte del tracto gastrointestinal que permanece unido a la pared abdominal dorsal. Evitar la ruptura de la tripa, ya que los contenidos pueden recubrir la pared abdominal y dificultan visualización del vaso dorsal. </li></ol><img alt="figura 3" src="/files/ftp_upload/55029/55029fig3.jpg" /> Figura 3: Vista del vaso dorsal. Una vez que el intestino y aguijón se han eliminado, el vaso dorsal es visible a lo largo de la línea media de la pared abdominal dorsal diseccionado. Barra de escala = 1 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55029/55029fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <ol start="5"><li> Organizar la pared abdominal dorsal en la orientación deseada por debajo de la cámara para que el vaso dorsal es visible (véase la Figura 3). Utilizar las tijeras de microdisección para recortar cualquier exceso de abdominal pared que impide la visualización del vaso dorsal. La forma de la pared abdominal dorsal debe ser similar a una taza o recipiente poco profundo cuando situada correctamente. <ol><li> Desde el vaso dorsal no se extiende en el segmento abdominal retaguardia de la abeja, quitar el Tergito final con el fin de mejorar la visualización del vaso dorsal. </li></ol></li><li> Utilizando una micropipeta de volumen ajustable, cubrir el vaso dorsal con 10 l de una solución isotónica para mantener las condiciones fisiológicas y facilitar un constante latido del corazón. NOTA: La solución recomendada es la fuerza trimestre solución de Ringer (0,120 g / l de cloruro de calcio, 0.105 g / L de cloruro de potasio, 0,050 g / L de bicarbonato de sodio, y 2,250 g / L de cloruro de sodio), que se ha encontrado para facilitar un establo, latido continuo. </li></ol> 3. La observación y la modulación del ritmo cardíaco <ol><li> Permitir que el vaso dorsal permanezca inmóvil hasta que se logre un pulso continuo estable(Normalmente dentro de 300 segundos). NOTA: El latido del corazón es visualizado como contracciones rítmicas del vaso dorsal. Inicialmente, puede parecer que no hay latido del corazón, especialmente si la abeja fue anestesiado, pero el corazón latiendo generalmente reanudar después de descansar en solución isotónica durante varios minutos y puede seguir batiendo durante horas, siempre que sigan siendo bañado en solución. </li><li> Medir la frecuencia cardíaca en términos del número de latidos por minuto (BPM). <ol><li> Registrar el número de contracciones observadas durante un periodo de 60 seg. Utilizar un contador de cuenta de la mano y un temporizador para facilitar este proceso. </li></ol></li><li> Medir el cambio en la frecuencia cardíaca mediante el registro de los BPM observado antes y después del tratamiento con un compuesto cardiomodulatory. NOTA: Aunque el tiempo necesario para observar un efecto sobre la frecuencia cardíaca puede variar dependiendo del compuesto que se está probando, los cambios en la frecuencia cardíaca normalmente se pueden observar dentro de minutos. <ol><li> Determinar la frecuencia cardíaca basal inmediatamente antes dela adición de cualquier compuesto de prueba. NOTA: la frecuencia cardiaca post-tratamiento por lo general se puede determinar después de 90 a 120 seg. </li><li> Preparar potenciales cardiomodulators (por ejemplo, octopamina) disolviendo el compuesto en la misma solución isotónica usada para bañar al vaso dorsal. </li><li> Añadir los compuestos de ensayo a la solución que rodea el vaso dorsal mediante la utilización de una micropipeta. </li></ol></li><li> Para una mayor precisión y un mayor rendimiento, una grabación de vídeo de cada sujeto de prueba y luego usar los videos para marcar el ritmo cardíaco en un momento posterior. NOTA: Esto permite que un solo investigador escalonar disecciones con el fin de facilitar la producción casi continua de videos. Cuando la grabación de vídeos, la longitud mínima recomendada es de aproximadamente 240 segundos con cualquier compuesto de ensayo que se añade en la marca de 60 seg. Esto asegura que el investigador tiene una ventana de 60 seg para asegurar la frecuencia cardíaca basal y luego otra ventana 60 seg para asegurar la frecuencia cardíaca post-tratamiento 120 secdespués del tratamiento. </li></ol>

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	<li>Klowden, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+Systems.">Circulatory Systems.</a> Physiological Systems in Insects, 3rd Edition. , 365-413 (2013).</li><li>League, G. P., Onuh, O. C., Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structural+and+functional+analysis+of+the+larval+and+adult+dorsal+vessel+and+its+role+in+hemolymph+circulation+in+the+mosquito+Anopheles+gambiae.">Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae.</a> J Exp Biol. 218 (Pt 3), 370-380 (2015).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+Hemolymph+Circulation+in+the+Butterfly+Papilio-Machaon+L+Revealed+by+Contact+Thermography+and+Photocell+Measurements.">Oscillating Hemolymph Circulation in the Butterfly Papilio-Machaon L Revealed by Contact Thermography and Photocell Measurements.</a> J Comp Physiol. 139 (2), 145-163 (1980).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+circulation+and+tracheal+ventilation+in+holometabolous+insects.">Interaction of circulation and tracheal ventilation in holometabolous insects.</a> Adv Insect Physiol. 26, 297-351 (1996).</li><li>Schwab, E. R., Chilson, R. A., Eddleman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heartbeat+Rate+Modulation+Mediated+by+the+Ventral+Nerve+Cord+in+the+Honey-Bee,+Apis-Mellifera.">Heartbeat Rate Modulation Mediated by the Ventral Nerve Cord in the Honey-Bee, Apis-Mellifera.</a> J Comp Physiol B-Biochem Syst Environ Physiol. 161 (6), 602-610 (1991).</li><li>Kaiser, W., Weber, T., Otto, D., Miroschnikow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+supply+of+the+heart+and+electrocardiogram+potentials+with+reversed+polarity+in+sleeping+and+resting+honey+bees.">Oxygen supply of the heart and electrocardiogram potentials with reversed polarity in sleeping and resting honey bees.</a> Apidologie. 45 (1), 73-87 (2014).</li><li>Papaefthimiou, C., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine--a+single+modulator+with+double+action+on+the+heart+of+two+insect+species+(Apis+mellifera+macedonica+and+Bactrocera+oleae):+Acceleration+vs.+inhibition.">Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.</a> J Insect Physiol. 57 (2), 316-325 (2011).</li><li>Castro, C. A., Hogan, J. B., Benson, K. A., Shehata, C. W., Landauer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral-Effects+of+Vehicles+-+Dmso,+Ethanol,+Tween-20,+Tween-80,+and+Emulphor-620.">Behavioral-Effects of Vehicles - Dmso, Ethanol, Tween-20, Tween-80, and Emulphor-620.</a> Pharmacol Biochem Behav. 50 (4), 521-526 (1995).</li><li>Papaefthimiou, C., Papachristoforou, A., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+responses+of+the+honeybee+heart+to+nanomolar+concentrations+of+amitraz.">Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz.</a> Pestic Biochem Phys. 107 (1), 132-137 (2013).</li><li>Roeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+in+invertebrates.">Octopamine in invertebrates.</a> Prog Neurobiol. 59 (5), 533-561 (1999).</li><li>Johnson, E., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Drosophila+heartbeat+by+neurotransmitters.">Modulation of Drosophila heartbeat by neurotransmitters.</a> J Comp Physiol B. 167 (2), 89-97 (1997).</li></ol>

The authors have nothing to disclose.

The protocol presented here provides a simple and effective approach to testing pharmacological compounds for their effects on honey bee heart rate. As observed in prior experiments that either transect the ventral nerve cord of a living insect5 or dissect out the ventral nerve cord when exposing the dorsal vessel7, the loss of central nervous system regulation results in a stable, low frequency heartbeat. The low frequency of beats allows the investigator to visually assess heart rate without havin...

El objetivo general de esta metodología es permitir que el investigador para observar y cuantificar el efecto que tiene un agente farmacológico en el ritmo cardíaco de las abejas melíferas forma rápida y sencilla. Las abejas, como otros insectos, tienen un sistema circulatorio abierto que difunde hemolinfa, el equivalente de insectos de la sangre, a través de la cavidad del cuerpo, conocida como la hemocoel. La circulación de hemolinfa es esencial para el transporte de nutrientes, factores inmunológicos, productos de desecho, así neurohormonas y otras moléculas de señalización 1. Circulation es facilitado por el vaso dorsal, que se extiende a lo largo de la línea media dorsal del insecto, así como los órganos pulsátiles accesorios. El vaso dorsal se divide en dos secciones funcionalmente distintas, designado el corazón en el abdomen y la aorta en el tórax y la cabeza. contracciones propagadas en la hemolinfa corazón bombee hacia el tórax y la cabeza, mientras que los órganos pulsátiles accesorios garantizan el flujo de la hemolinfa de las extremidades. <pclass = &quot;jove_content&quot;&gt; función cardíaca de insectos se puede observar usando una variedad de métodos, dependiendo de la magnitud, fase fisiología, o la vida del insecto. Un enfoque común para la observación de la frecuencia cardíaca en larvas o insectos más pequeños es el uso de imágenes intravital 2. Este método es menos útil en las abejas adultas, sin embargo, ya que puede ser difícil de ver claramente el vaso dorsal a través de la pared abdominal. Un enfoque establecido para el registro de la frecuencia cardíaca en una variedad de insectos, incluyendo abejas, es el uso de la termografía de contacto, que utiliza termistores aplicado en el exterior del insecto para detectar pulsaciones cardiacas 3,4. La frecuencia cardíaca en las abejas adultas también ha sido grabado utilizando una técnica electrofisiológica para medir una señal de impedancia eléctrica de 4,5. Esta técnica requiere la inserción de electrodos en el animal al lado del corazón y el uso de un convertidor de impedancia para registrar los latidos del corazón 4. Del mismo modo, electrocardiogramas se han utilizado para DETECt señales eléctricas producidas por el corazón y combinados con grabación de vídeo de la abeja para observar los cambios en la actividad cardíaca 6. Una clara ventaja de estos enfoques es que la frecuencia cardíaca se evalúa en una, abeja vivas intactas, en lugar de en un espécimen disecado, que ayuda a asegurar la disponibilidad de toda la gama de respuestas fisiológicas en el tema. Los desafíos de estos enfoques incluyen la contabilidad para la inmovilización o la anestesia de la materia, la necesidad de limitar las variables externas y estímulos que podrían alterar la frecuencia cardiaca, así como la determinación de un método de entrega adecuado al probar agentes farmacológicos. Otro enfoque que se ha utilizado para el estudio de la actividad cardiaca de abeja es para diseccionar parcialmente el insecto con el fin de exponer el corazón, a continuación, medir contracciones vaso dorsal utilizando un transductor de desplazamiento de fuerza 7. En este protocolo, el corazón se baña continuamente con solución salina fisiológica en marcha y prueba de compounds se pueden disolver en esta solución para su aplicación al objeto 7. Una diferencia significativa entre este método y las descritas anteriormente es que el cordón nervioso ventral se elimina, eliminando el papel que el sistema nervioso central se ha demostrado que desempeña en la modulación de la frecuencia cardíaca 5. El resultado es que el latido del corazón de referencia, que suele ser bastante irregular, se estabiliza a una frecuencia mucho más baja y amplitud que se observa típicamente en un insecto que viven 5,7. Lo que todos estos métodos tienen en común es que requieren un equipo altamente especializado y, a menudo caro, además de un cierto nivel de experiencia, con el fin de llevar a cabo. Tal vez la mayor desventaja es que ninguno de estos enfoques son particularmente adecuados para experimentos que involucran el examen de un gran número de temas, tales como selección de una biblioteca de compuestos potencialmente cardiomodulatory. La mayor fortaleza del método descrito aquíes su simplicidad. El protocolo es relativamente fácil de dominar, la instalación requiere poco espacio, y sólo una entrada financiero mínimo es necesario. El método requiere poco más que algunas abejas, algunos instrumentos quirúrgicos, una solución isotónica, y, o bien un microscopio digital o un microscopio tradicional con una cámara digital. Las abejas son diseccionados para visualizar el vaso dorsal y vídeos digitales se utilizan para registrar la frecuencia cardíaca antes y después del tratamiento con agentes farmacológicos. Aunque la grabación de vídeo no es realmente necesario para observar los cambios en el ritmo cardíaco, además de aumentar considerablemente el rendimiento (es decir, el número de sujetos que se pueden procesar en un período de tiempo determinado). El investigador puede maximizar la eficiencia mediante el registro de un gran número de vídeos a la vez y, posteriormente, marcando estos videos en un momento más conveniente. Otra ventaja de este enfoque es que los vídeos permiten al investigador a empezar de nuevo, se debe interrumpir el proceso de puntuación, y que sea más fácil para el vdel Visor para cegar al tratamiento con el fin de reducir el sesgo.

<table><tbody><tr><td>Dino-Lite Edge digital USB microscope</td><td>Dino-Lite</td><td>AM4815ZT</td><td>Any digital microscope or similar setup will suffice</td></tr><tr><td>Microscope stand</td><td>Dino-Lite</td><td>RK-10</td><td>Any stand appropriate for the digital microscope</td></tr><tr><td>Laptop or PC</td><td></td><td></td><td>Necessary for digital microscope</td></tr><tr><td>Microdissection scissors (Vannas, 8 cm, Straight, 5 mm Blades)</td><td>World Precision Instruments</td><td>14003</td><td>Any similar scissors suitable for microdissection will suffice</td></tr><tr><td>Microdissecting Forceps, 10.2 cm, Angled (2 pair)</td><td>World Precision Instruments</td><td>504482</td><td>Any similar forceps suitable for microdissection will suffice</td></tr><tr><td>Ringers solution 1/4 strength tablets&amp;nbsp;</td><td>Sigma-Aldrich</td><td>96724-100TAB</td><td></td></tr><tr><td>Dissecting tray</td><td></td><td></td><td>Any surface suitable for microdissection</td></tr><tr><td>Single channel 10 &amp;micro;l pipette</td><td></td><td></td><td>Any device capable of accurately delivering 10 &amp;micro;l volume</td></tr><tr><td>Pipette tips</td><td></td><td></td><td></td></tr><tr><td>Small beaker or container of water</td><td></td><td></td><td>Used to rinse instruments between subjects</td></tr><tr><td>Hand tally counter</td><td>Office Depot</td><td>295033</td><td>Any similar product will suffice</td></tr><tr><td>Timer</td><td>Office Depot</td><td>644219</td><td>Any similar product will suffice</td></tr><tr><td>Deionized water</td><td></td><td></td><td>Preparation of Ringers solution and rinsing instruments</td></tr></tbody></table>

dissection and observation of honey bee dorsal vessel for studies of cardiac function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

Dado que muchos de los compuestos farmacológicamente activos que pueden ser probados usando este protocolo no son solubles en agua, es necesario tener un disolvente fiable que permitirá a los compuestos de ensayo para ser entregados a través de la solución isotónica usada para bañar al vaso dorsal. Dimetil sulfóxido (DMSO) es un disolvente que se utiliza comúnmente como un vehículo para la administración de fármacos experimentales y otros compuestos en los animales 8,</su...

Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

La disección y la observación de la abeja de la miel dorsal del recipiente de Estudios de la función cardiaca

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

dissection-observation-honey-bee-dorsal-vessel-for-studies-cardiac

Research

JoVE Journal

Biology

10.8K vistas.  Virginia Tech. The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

Video: Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Análisis de los vasos coronarios en los corazones embrionarias aclarados

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Investigación

Biología del Desarrollo

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

La visualización de las variaciones del ciclo celular y la determinación de la nucleación en cardiomiocitos postnatales

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualizar el corazón latiendo en Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Biología

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Monitorización de la función del corazón en las larvas Drosophila melanogaster Para los estudios fisiológicos

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and&#160;achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

The Drosophila heart sectioning and fluorescence imaging protocol simplifies studying heart structure and pathologies. This approach involves straightforward sectioning, staining, and imaging, bypassing the technical expertise needed for traditional dissection. It enhances accessibility, making Drosophila a more widely usable model for cardiac-related research within the broader scientific community.

Método novedoso y eficiente para la tinción de fluorescencia cardíaca de Drosophila con criosección

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila ...

Genética

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

La disección del corazón en las larvas, juveniles y adultos de pez cebra, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Roedor de Trabajo Modelo del corazón para el estudio sobre los resultados de miocardio y el consumo de oxígeno

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

Técnicas de disección y muestreo histológico del corazón en grandes modelos animales para enfermedades cardiovasculares

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Medicina

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparación e Imagen longitudinal de la función cardiaca in vivo utilizando microscopía de coherencia óptica (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Heart Dissection: A Method to Observe Zebrafish Cardiac Development

Source: Singleman C. et. al., <a href="https://www.jove.com/v/3165/" target="_blank">Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio</a>. J. Vis. Exp. (2011).
This video describes a technique of isolating the heart from a zebrafish at multiple developmental stages to study cardiac development, maturation, and ageing.

Disección del corazón: un método para observar el desarrollo cardíaco del pez cebra

Source: Singleman C. et. al., Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio. J. Vis. Exp. (2011).
This video describes a technique ...

La disección y la observación de la abeja de la miel dorsal del recipiente de Estudios de la función cardiaca

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