Los autores desean agradecer a Laura Díaz, Kevin Phillips, Willa A. Hsueh, y el Rey C. Li por su apoyo útiles comentarios y técnico en el desarrollo de este método.

1. Animal Preparación y Tratamiento de Frío <ol><li> Localizar e inspeccionar un 4 ° C habitación fría que ha sido aprobado para el alojamiento de los ratones de laboratorio. </li><li> Pre-frialdad animales durante la noche en el cuarto frío jaulas. Las jaulas se montan sin alimentación y las camas pero con una botella de agua. </li><li> En la mañana del día experimental, los ratones lugar de uno en uno en cada una de las jaulas de pre-refrigerado a intervalos de 30 min. Cada ratón enjaulado individualmente deben permanecer en la sala fría durante casi 4 horas antes de su transporte al laboratorio de imagen. Asegúrese de que los ratones son el ayuno, pero con acceso al agua. </li><li> A las 4 h después de transporte tratamiento en frío un animal a la vez cada 30 min para el laboratorio de imagen. Esto se puede lograr por llenado de un recipiente de espuma de poliestireno con hielo y la colocación de una jaula de alojamiento previamente enfriado en la parte superior del hielo dentro de la caja. Libremente colocar la tapa de la caja de espuma de poliestireno. </li></ol> 2. Configuración de flujo de trabajo de imágenes Micro-PET/CT <p class=&quot;Jove_content&quot;&gt; En esta imagen de protocolo micro-PET/CT se consigue con la Siemens Inveon Dedicated PET (DPET) Sistema y multimodalidad Inveon (mm) Sistema (CT / SPECT) en el modo acoplado. El animal se coloca desde la entrada MM, primero se explora con la CT de referencias anatómicas, y luego se deslizó hacia el centro de la DPET para una adquisición PET estática F18. A fin de que el ordenador central para llevar a cabo estas tareas secuenciales de forma automática, la siguiente &quot;flujo de trabajo&quot; se programa con (IAW) el lugar de trabajo Adquisición Inveon software antes de la sesión de imagen real. <ol><li> CT adquisición: Para una tomografía computarizada de cuerpo entero, ajuste de corriente a 500 uA, la tensión en 80kV, tiempo de exposición de 200 ms y 240 pasos de 240 ° de rotación. Para detector de rayos X, seleccione Resolución en el &quot;sistema de baja magnificación&quot; con 78 mm de campo de imagen y el modo axial cama individual. Seleccione &quot;reconstrucción en tiempo real&quot; a través del &quot;Common Cone-Beam Reconstrucción&quot; método para que las conversaciones de PC host conun dedicado equipo de reconstrucción en tiempo real (Cobra) para iniciar la tarea. </li><li> PET Adquisición de emisiones: Set 600 segundos (10 minutos) para el &quot;tiempo de ciclo fijo&quot; en el &quot;adquirir por el tiempo&quot; opción. Seleccione F-18 como &quot;el estudio de isótopos&quot; y utilizar 350-650 keV como &quot;nivel de energía&quot;. </li><li> PET Histograma de emisiones: Set &quot;marco dinámico&quot; como &quot;negro&quot; para procesar los datos como un marco para toda la duración de lograr exploración estática. Seleccione &quot;3D&quot; como el tipo de histograma y elija &quot;sin corrección de dispersión&quot;. </li><li> PET Reconstrucción: Use 2D Maximización Pedido Expectativa Subset (OSEM2D) como algoritmo de reconstrucción. </li></ol> 3. La inyección de FDG <ol><li> Solicite un paquete clínico de 18 F-FDG (10 mCi) de un proveedor regional de su llegada al laboratorio de imagen ~ 30 min antes de la primera inyección programada. Siga los procedimientos del instituto de seguridad para recibir y examinar el paquete que contiene materia radiactivals (RAM). </li><li> Con la protección que ofrece un escudo de mesa L-bloque superior, la alícuota FDG y hacer diluciones con solución salina esterilizada. La concentración de actividad de la FDG diluida debe estar disponible a 200-300 μCi/100 ul para cada inyección. Dibujar la solución FDG en una jeringa de 1 ml con 26G 1/2 pulgada aguja, y medir la radiactividad de la jeringa entera con un calibrador de dosis. </li><li> Inyectar al animal que es transportado desde la sala de frío (véase el Paso 1,4) con 100 l de solución de FDG a través de la vía intraperitoneal (ip). Registre el tiempo de inyección. Medir la radiactividad residuo de la jeringa de nuevo con el calibrador de dosis. </li><li> Ponga el animal a la jaula frío dentro de una hielera de unicel mantenido con hielo. Incubar el animal en frío (~ 4 ° C) durante 1 hora para la captación de FDG. </li><li> Cálculo de la actividad inyectada FDG para cada ratón mediante la siguiente fórmula: Actividad inyectada (Ci) = actividad en la jeringa antes de la inyección- Actividad en la jeringa después de la inyección </li></ol> 4. Micro-PET/CT imágenes <ol><li> Las imágenes micro-PET/CT comienza 1 hora después de la inyección de FDG o 5 horas después del tratamiento en frío. Ponga el animal en una cámara de inducción de la anestesia con isoflurano 3% de oxígeno. </li><li> Una vez que se induce la anestesia, el animal se mueve sobre una pastilla de micro-CT (cama de animales) con su cabeza descansando dentro de una máscara de cara cono que suministra continuamente isoflurano (2%) a un caudal de 2 L / min. Una almohadilla eléctrica de calentamiento (Biovet, m2m Imaging Corp.) se coloca bajo el animal, para ayudar a mantener la temperatura del cuerpo. </li><li> Deslice el animal a la entrada del escáner MM, active el &quot;láser&quot; icono de la barra de herramientas y utilice el control de panel táctil para mover la cama para que el pecho del animal es en la cruz de líneas láser horizontales y verticales. En el &quot;Laser Align&quot; ventana, seleccione &quot;Tipo de primer ciclo&quot;, como tomografía computarizada, y &quot;adquisición PET incluido en el flujo de trabajo&quot;, como el option. </li><li> Abra la ventana &quot;Ver Scout&quot; ventana y adquirir una visión explorador de rayos X radiografía. Use IAW para ajustar la posición de la cama animal de modo que el centro del campo de vista de CT se encuentra en el centro del cuerpo del ratón (hígado). Repita este paso si es necesario. </li><li> Inicie el &quot;flujo de trabajo&quot; establecido en el Paso 2. Cuando las opciones de pop-up, seleccionar un rango de 3D PET-CT archivo de la matriz de transformación que se utilizarán en la reconstrucción de CT, y elegir un archivo de normalización recientemente creado para la reconstrucción de PET sin corrección de atenuación. El IAW continuación, se iniciará la TC y PET de forma secuencial según se haya programado. </li><li> Después de que el flujo de trabajo conjunto se completa, que puede tomar 20 a 25 min, brevemente evaluar la calidad de la adquirieron imágenes de CT y PET con el VM ASIPro, un software de análisis de micro-PET co-instalado con IAW. Archivo de los datos de imagen en un dispositivo de almacenamiento de datos o transferir los datos a través de la red a una estación de trabajo de análisis de imágenes post (ver paso 5) para su posterior análisis. </li><li> Releasí al animal de los sistemas de imagen y colocarlo a una jaula de alojamiento limpio y con comida normal y suministro de agua para su recuperación a temperatura ambiente. Los sistemas están ahora listos para el animal siguiente en la cola. Tenga en cuenta el cuidado y manejo de animales de imágenes post-deben seguir los reglamentos del Instituto en relación con &quot;el manejo de animales de laboratorio inyectados con RAM&quot;. También tenga en cuenta que las agujas / jeringas, las almohadillas absorbentes, guantes y ropa de cama de todo y la materia fecal debe ser considerado como residuo radiactivo, y manejados de acuerdo a los reglamentos pertinentes del instituto RAM eliminación de residuos. </li></ol> 5. Post-Análisis de imágenes <ol><li> Open Research Inveon lugar de trabajo (CPIC) software (Siemens) y manualmente importar tanto la TC y PET conjuntos de datos. Co-registro de imágenes de TC y PET en &quot;Registro&quot; de la ventana usando herramientas con el &quot;análisis general&quot; la función, y en la sección &quot;Opinión&quot; ventana asegurarse una perfecta alineación entre la TC y las imágenes de PET en las 3 dimensiones. </li><li>De &quot;zona de interés (ROI) La cuantificación&quot; de la ventana, con las referencias proporcionadas por las imágenes co-registradas CT, identificar BAT en la región interescapular del cuello, la más predominante inducible por frío BAT en ratones adultos, y dibujar volumen de interés (VOI) de BAT sobre el conjunto de datos de PET. Seleccione &quot;Intensidad Voxel&quot; como &quot;Tipo de cuantificación&quot; y registrar la radiactividad en el medio VOI como Bq / ml. Un factor de calibración que convierte cuentas / seg a Bq / ml ha sido previamente establecido mediante el escaneo de un fantasma con radioactividad conocida. </li><li> La captación de FDG en la MTD se cuantifica como porcentaje de dosis inyectada por gramo de tejido (% ID / g) con la corrección de la descomposición. La dosis inyectada es el resultado del paso 3,5, sin embargo, se convirtió al becquerel (Bq) unidad (1 Ci = 37.000 Bq); suponemos que 1 ml de tejido es igual a 1 g. </li></ol>

<ol>
	<li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+importance+of+brown+adipose+tissue+in+energy+homeostasis.">The role and importance of brown adipose tissue in energy homeostasis.</a> Curr. Opin. Pediatr. 22, 478-484 (2010).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiol. Rev. 84, 277-359 (2004).</li><li>Collins, S., Surwit, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-adrenergic+receptors+and+the+control+of+adipose+tissue+metabolism+and+thermogenesis.">The beta-adrenergic receptors and the control of adipose tissue metabolism and thermogenesis.</a> Recent Prog. Horm. Res. 56, 309-328 (2001).</li><li>Ouellet, V., 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=Outdoor+temperature,+age,+sex,+body+mass+index,+and+diabetic+status+determine+the+prevalence,+mass,+and+glucose-uptake+activity+of+18F-FDG-detected+BAT+in+humans.">Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans.</a> J. Clin. Endocrinol. Metab. 96, 1115-1125 (2011).</li><li>Cypess, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> N. Engl. J. Med. 360, 1509-1517 (2009).</li><li>Fruhbeck, G., Becerril, S., Sainz, N., Garrastachu, P., Garcia-Velloso, 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=BAT:+a+new+target+for+human+obesity.">BAT: a new target for human obesity.</a> Trends Pharmacol. Sci. 30, 387-396 (2009).</li><li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+fat+as+a+therapy+for+obesity+and+diabetes.">Brown fat as a therapy for obesity and diabetes.</a> Curr. Opin. Endocrinol. Diabetes Obes. 17, 143-149 (2010).</li><li>Virtanen, K. A., 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=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> N. Engl. J. Med. 360, 1518-1525 (2009).</li><li>van Marken Lichtenbelt, W. D., 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=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> N. Engl. J. Med. 360, 1500-1508 (2009).</li><li>Cohade, C., Mourtzikos, K. A., Wahl, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="USA-Fat":+prevalence+is+related+to+ambient+outdoor+temperature-evaluation+with+18F-FDG+PET/CT.">"USA-Fat": prevalence is related to ambient outdoor temperature-evaluation with 18F-FDG PET/CT.</a> J. Nucl. Med. 44, 1267-1270 (2003).</li><li>Garcia, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+brown+fat+2-deoxy-2-[F-18]fluoro-D-glucose+uptake+by+controlling+environmental+temperature+prior+to+positron+emission+tomography+scan.">Reduction of brown fat 2-deoxy-2-[F-18]fluoro-D-glucose uptake by controlling environmental temperature prior to positron emission tomography scan.</a> Mol. Imaging Biol. 8, 24-29 (2006).</li><li>Saito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58, 1526-1531 (2009).</li><li>Agrawal, A., Nair, N., Baghel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+reduction+of+brown+fat+uptake+on+FDG+PET.">A novel approach for reduction of brown fat uptake on FDG PET.</a> Br. J. Radiol. 82, 626-631 (2009).</li><li>Soderlund, V., Larsson, S. A., Jacobsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+FDG+uptake+in+brown+adipose+tissue+in+clinical+patients+by+a+single+dose+of+propranolol.">Reduction of FDG uptake in brown adipose tissue in clinical patients by a single dose of propranolol.</a> Eur. J. Nucl. Med. Mol. Imaging. 34, 1018-1022 (2007).</li><li>Zhao, J., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenesis+is+beta3-+but+not+beta1-adrenergically+mediated+in+rat+brown+fat+cells,+even+after+cold+acclimation.">Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.</a> Am. J. Physiol. 275, 2002-2011 (1998).</li><li>Rowland, D. J., Garbow, J. R., Laforest, R., Snyder, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+[18F]FDG+microPET+and+small-animal+MRI.">Registration of [18F]FDG microPET and small-animal MRI.</a> Nucl. Med. Biol. 32, 567-5672 (2005).</li></ol>

No hay conflictos de interés declarado.

En este estudio un método de imagen micro-PET/CT-based ha sido desarrollado para la detección de actividades de BAT en ratones adultos que simplemente se requiere un tratamiento de frío y una inyección de disponible en el mercado con 18 F-FDG. El procedimiento completo se puede realizar en un día después de un tratamiento y la secuencia de formación de imágenes que se inicia cada minuto 30 hasta que todos los animales son tratados y fotografiado. En las condiciones experimentales descritas, un total d...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Micro-PET/CT Imaging System </td><td> Siemens Medical Solutions EE.UU., Inc. </td><td> Inveon dedicado PET Sistema y Inveon multimodalidad CT / SPECT del sistema (acoplado) </td><td></td></tr><tr><td> Propranolol </td><td> Sigma </td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td> Sigma </td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope Inc. </td><td></td><td></td></tr><tr><td> C57BL/6J ratones macho </td><td> Jackson Laboratory </td><td> 000664 </td><td> 3-4 meses de edad </td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

Tejido adiposo marrón (BAT) difiere de tejido adiposo blanco (WAT) por su ubicación discreta y un color marrón-rojo debido a la vascularización rico y de alta densidad de las mitocondrias. BAT juega un papel importante en el gasto energético y no tiritando termogénesis en los mamíferos recién nacidos, así como los adultos 1. BAT-mediada por la termogénesis está altamente regulada por el sistema nervioso simpático, principalmente por medio del receptor adrenérgico β 2, 3. Estudios recientes han demostrado que las actividades de BAT en humanos adultos tienen una correlación negativa con el índice de masa corporal (IMC) y otros parámetros diabéticos 4-6. BAT Así, se ha propuesto como un objetivo potencial para la terapia anti-obesity/anti-diabetes centrándose en la modulación del balance energético de 6-8. Mientras que varios frías a base de desafío-tomografía por emisión de positrones (PET) se establecen los métodos para la detección de BAT humano 9-13, esencialmente no hay protocolo estandarizado para la obtención de imágenes y quantificación de BAT en modelos de animales pequeños tales como ratones. Aquí se describe un robusto PET / CT método de imagen para la evaluación funcional de las MTD en ratones. En pocas palabras, los ratones C57BL/6J adultos estaban fríos tratados bajo condiciones de ayuno con una duración de 4 horas antes de recibir una dosis de 18 F-fluorodesoxiglucosa (FDG). Los ratones se mantuvo en el frío por un puesto adicional de inyección de FDG horas, y luego escaneadas con un sistema micro-PET/CT pequeño animal dedicado. Las imágenes de PET adquiridas fueron co-registradas con las imágenes de TC para las referencias anatómicas y se analizó la captación de FDG en el área interescapular BAT para presentar la actividad de las MTD. Esta estandarizado en frío y tratamiento de protocolo de imágenes ha sido validado a través de actividades de prueba BAT durante las intervenciones farmacológicas, por ejemplo, la activación BAT suprimida por el tratamiento de la β-adrenérgico propranolol 14, 15, o la activación BAT mejorada por β3 agonista BRL37344 16. El método described aquí se puede aplicar para la detección de fármacos / compuestos que modulan la actividad de BAT, o para identificar los genes / vías que están involucrados en el desarrollo y regulación de BAT en diversos estudios preclínicos y de base.

Un ejemplo de formación de imágenes micro-PET/CT de BAT ratón se muestra en la figura 1. Si bien la formación de imágenes CT proporciona información anatómica, la formación de imágenes PET codifica la distribución y la cantidad de 18 F-FDG por todo el cuerpo. Estos datos de imágenes se pueden ver por separado (1A y 1B), fusionado (1C), o resultar de una característica de 3D, como la proyección de intensidad máxima (MIP, 1D). Con la ayuda de una herramienta de formación de imág...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Un método de obtención de imágenes funcionales de tejido de ratón adiposo marrón (BAT) se describe en frío que estimula la captación de 18F-fluorodeoxiglucosa (FDG) en la MTD es evaluar de forma no invasiva con un protocolo estandarizado micro-PET/CT. Este método es robusto y sensible para detectar diferencias en las actividades de BAT en modelos de ratón.

Imagen funcional de grasa marrón en ratones con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K vistas.  The Methodist Hospital Research Institute, Houston. Un método de obtención de imágenes funcionales de tejido de ratón adiposo marrón (BAT) se describe en frío que estimula la captación de 18F-fluorodeoxiglucosa (FDG) en la MTD es evaluar de forma no invasiva con un protocolo estandarizado micro-PET/CT. Este método es robusto y sensible para detectar diferencias en las actividades de BAT en modelos de ratón.

Video: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Luz / oscuridad de transición de prueba para ratones

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Investigación

Biología

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etiquetado y hESCs hMSCs con nanopartículas de óxido de hierro para no invasivo en el seguimiento in vivo mediante RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Imágenes funcionales con Refuerzo, Eyetracking y Monitoreo Fisiológico

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Laberinto elevado Plus para ratones

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Concentración, la carga, y la alineación de una célula de ultracentrifugación analítica de muestras

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de Imagen Ca 2 + Señalización en el esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

Un método de alternante a la modelo tradicional de la NASA Miembro Posterior descarga en ratones

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Mapeo de las secuelas de la theta ráfaga de estimulación sobre la corteza auditiva humana con imágenes funcionales

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imágenes ópticas basadas en fotodiodos para registrar dinámicas de red con resolución de una sola neurona en invertebrados no transgénicos

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...