Los autores agradecen a Voltaire Gungab de asistencia con la preparación del manuscrito. Este trabajo fue apoyado en parte por subvenciones de: los Institutos Nacionales de Salud (T32 GM008440 a EJ Walker, R01 NS27713 a WL Young, P01 NS44155 a WL jóvenes y Su H.), y el R21 NS070153 de H. Su, y el americano Heart Association (AHA 10GRNT3130004 de H. Su).

1. Limpieza de sangre de la vasculatura <ol><li> Anestesiar a ratones mediante la inyección intraperitoneal de 100 mg / kg de ketamina con 10 mg / kg de xilazina diluida en solución salina 0,25 ml. </li><li> Una vez que la anestesia ha sido confirmado por no responder a una pizca de la pata, utilizar tijeras para hacer una incisión en el pecho con unas tijeras justo debajo de la apófisis xifoides, corte a través del diafragma, y ​​cortada entre el segmento dorsal y ventral de la caja torácica para exponer la cavidad torácica. Exponer el corazón doblando el esternón y la pared torácica adyacente y seguro con pinzas hemostáticas. Abrir el saco pericárdico con pinzas para exponer el corazón. </li><li> Encienda la bomba de perfusión (100 mmHg de presión) conectado a PBS caliente más heparina (5 unidades / ml) en un baño de agua caliente a 37 ° C y el tubo con una aguja de calibre 22 en la contundente salida. </li><li> Insertar la aguja en el ventrículo izquierdo, cerca del ápice del corazón. Inmediatamente abrieron la aurícula derecha para permitir flujo arterial sistémica y continuar el lavado hasta la sangre is retiró de la circulación. </li></ol> 2. La perfusión de agente de casting en el sistema vascular <ol><li> Prepare Microfil (Flow Tech, Inc. Carver, MA) solución de fundición por el protocolo del fabricante: Mezclar 5 ml de diluyente MV con 4 ml de compuesto MV filtrado. Agregar 450μl (5%) de catalizador (MV agente de curado). Tiempo de solución de trabajo es de 20 minutos, pero la mezcla inmediatamente antes de su uso para asegurar la viscosidad adecuada. </li><li> Retirar de fundición solución del agente en una jeringa de 10 ml, adjuntar un contundente 20 aguja a la jeringa y la aguja de llenado con la solución. </li><li> Insertar la aguja en el ventrículo izquierdo a través del mismo agujero utilizado para la irrigación de la sangre. Aguja asegura en su lugar con la pinza hemostática en el corazón. </li><li> Inyectar la solución de fundición agente lentamente hacia el ventrículo izquierdo a aproximadamente 3 ml / min. El exceso de presión puede conducir a un flujo incorrecto de agente de casting o de ruptura de los vasos potencial. </li><li> Busque signos de perfusión con éxito, incluyendo: Visualización del repartoción del agente en la vasculatura intestino y el hígado, decoloración de la extremidad distal, y la nariz y la decoloración de la lengua. Reajustar la aguja, si es necesario para lograr una perfusión adecuada. </li></ol> 3. Recolección y procesamiento de tejidos <ol><li> Quitar el cerebro y el lugar en paraformaldehído al 4% durante la noche a 4 ° C. </li><li> Deshidratar el cerebro mediante la colocación de paraformaldehído directamente en EtOH concentra cada vez más a temperatura ambiente: 25% EtOH, 1 día, 50% de EtOH, 1 día, 75% de EtOH, un día, y el 95% de EtOH, 2 días, y el 100% EtOH, 2 días . </li><li> Aclarar el tejido cerebral alrededor de los vasos sanguíneos del cerebro mediante la inmersión de perfusión en salicilato de metilo durante 2 días a temperatura ambiente. </li><li> Vasos sanguíneos de todo el cerebro unsectioned aclarado se pueden visualizar con el cerebro inmerso en salicilato de metilo bajo un microscopio de disección (Fig. 1) (Leica MZFL microscopio III, Leica Microsystems, Bannockburn, IL) o microscopio de campo claro (Fig. 2) (Leica DM / LS, Leica Microsystems). </li><li> Reclarificationse puede hacer si el tejido no esté completamente curada. Del cerebro de la transferencia de salicilato de metilo, el 95% de EtOH durante 2 días, 100% EtOH durante 2 días, y salicilato de metilo durante 2 días. </li></ol> 4. Micro-TC o la preparación histológica de aplicación de tejidos <ol><li> El cerebro se pueden visualizar mediante micro-CT directamente después de la aclaración o después de la re-hidratación (Fig. 4). </li><li> Para preparar el tejido cerebral para el análisis histológico, rehidratar el cerebro a través de EtOH diluida en serie: EtOH al 100%, un día, y el 95% de EtOH, 1 día, 75% de EtOH, 1 día, 50% de EtOH, 2 días, 25% de EtOH, 2 días y almacenar en PBS. El cerebro entonces puede ser embebido en parafina utilizando un procedimiento estándar. </li></ol> 5. Resultados representante Después de fundición vascular, la cerebrovasculature se puede ver macroscópicamente en la superficie del cerebro (Fig. 1A). Del árbol vascular en el interior del parénquima se puede visualizar la siguiente aclaración (Fig. 1B). La estructura vascular detalladapueden ser reflejados y analizados bajo un microscopio de campo claro (Fig. 2), la disección de alcance (Fig. 3), y con micro-TC (Fig. 4). Mediante microscopía de campo claro, los vasos se pueden ver en diferentes planos focales sin seccionar el cerebro (Fig. 2A), así como microvasos individuales a mayores aumentos (Fig. 2B). Bajo un microscopio de disección, se puede ver toda la estructura vascular en un plano focal, lo que permite una mejor visualización de la estructura tridimensional de la cerebrovasculature (Fig. 3). Las imágenes tomadas con un microscopio de disección puede mostrar la morfología de los vasos grandes y vasos de todo el cerebro, pero carecen de los datos que aparecen en las imágenes tomadas con el microscopio de campo claro (Fig. 2B). Esta técnica nos ha permitido detectar embarcaciones irregulares en nuestro modelo de ratón experimental BAVM adulto 2. La Figura 5 muestra un ejemplo de las mismas embarcaciones irregulares detectados por micro-TC (Fig. 5) y el microscopio de campo claro (Fig. 5B). En conjunto, estos datos muestran que se puede analizar tque muestra lo mismo con las herramientas de imágenes múltiples después de la fundición vascular. <img alt="Figura 1" src="/files/ftp_upload/2958/2958fig1.jpg" /> Figura 1. Cerebro de ratón toda la nueva fundición vascular. Antes (A) y después (B) la clarificación salicilato de metilo solvente. Los buques pueden ser claramente visualizado en la superficie del cerebro (A) y en el procedimiento de parénquima siguiente aclaración (B). <img alt="Figura 2" src="/files/ftp_upload/2958/2958fig2.jpg" /> Figura 2. Microscopio de campo claro imágenes aclaró cerebro vascular fundido. El cerebro se pueden obtener imágenes en diferentes planos y en diferentes niveles de ampliación. Barra de escala 200μm (A) y 50 micras (B). <img alt="Figura 3" src="/files/ftp_upload/2958/2958fig3.jpg" /> Figura 3. Disección de imagen microscópica muestra a 1x ratón adulto cerebrovasculature todo el cerebro de una vista dorsal. &lt;img alt = &quot;Figura 4&quot; src = &quot;/ files/ftp_upload/2958/2958fig4.jpg&quot; /&gt; Figura 4. Micro-TC imagen de la misma de todo el cerebro del ratón adulto se muestra en la Figura 3 a partir de una vista dorsal. <img alt="Figura 5" src="/files/ftp_upload/2958/2958fig5.jpg" /> Figura 5. Aplicación del método de fundición vascular para detectar una malformación arteriovenosa cerebral. A) Micro-TAC de cerebro vascular fundido muestra ampliada de la región, irregular AVM-como los vasos en el hemisferio derecho de una vista ventral. Imagen ampliada de la región (caja blanca) se muestra a la derecha. B) Lo mismo que los vasos irregulares visualizaron bajo microscopio de campo claro con un aumento de 50x. Barra de escala 50 micras.

<ol>
	<li>Bolland, B. J., Kanczler, J. M., Dunlop, D. G., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+in+vivo+muCT+evaluation+of+neovascularisation+in+tissue+engineered+bone+constructs.">Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs.</a> Bone. 43, 195-202 (2008).</li><li>Walker, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriovenous+malformation+in+the+adult+mouse+brain+resembling+the+human+disease.">Arteriovenous malformation in the adult mouse brain resembling the human disease.</a> Ann. Neurol. , (2011).</li><li>Iqbal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+novel+mono-+and+multivalent+VHH-fragments+and+their+application+for+molecular+imaging+of+brain+tumours.">Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.</a> Br. J. Pharmacol. 160, 1016-1028 (2010).</li><li>Fukunaga, A., Kawase, T., Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+after+simultaneous+transplantation+with+neuro-epithelial+stem+cells+and+adjacent+mesenchymal+tissues+into+infarcted+rat.">Functional recovery after simultaneous transplantation with neuro-epithelial stem cells and adjacent mesenchymal tissues into infarcted rat.</a> 145, 473-480 (2003).</li><li>Chugh, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cerebral+blood+volume+in+mouse+brain+regions+using+micro-computed+tomography.">Measurement of cerebral blood volume in mouse brain regions using micro-computed tomography.</a> Neuroimage. 47, 1312-1318 (2009).</li><li>Marxen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Med. Phys. 31, 305-313 (2004).</li><li>Barger, A. C., Beeuwkes, R., 3rd, L. a. i. n. e. y., L, L., Silverman, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis:+vasa+vasorum+and+neovascularization+of+human+coronary+arteries.+A+possible+role+in+the+pathophysiology+of+atherosclerosis.">Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis.</a> N. Engl. J. Med. 310, 175-177 (1984).</li><li>Fossett, D. T., Caputy, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Operative Neurosurgical Anatomy. , (2002).</li><li>Reynolds, D. G., Brim, J., Sheehy, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+architecture+of+the+small+intestinal+mucosa+of+the+monkey+(Macaca+mulatta).">The vascular architecture of the small intestinal mucosa of the monkey (Macaca mulatta).</a> Anat. Rec. 159, 211-218 (1967).</li><li>Bernard, S., Luchtel, D. L., Polissar, N., Hlastala, M. P., Lakshminarayan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+size+of+bronchopulmonary+anastomoses+in+sheep+lung.">Structure and size of bronchopulmonary anastomoses in sheep lung.</a> Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 286, 804-813 (2005).</li><li>Brady, J. M., Cutright, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+of+measuring+blood+vessel+volume+in+bone+applied+to+the+mandible+and+humerus+of+the+rat.">A new technique of measuring blood vessel volume in bone applied to the mandible and humerus of the rat.</a> Anat. Rec. 170, 143-146 (1971).</li><li>Evan, A. P., Dail, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferent+arterioles+in+the+cortex+of+the+rat+kidney.">Efferent arterioles in the cortex of the rat kidney.</a> Anat. Rec. 187, 135-145 (1977).</li><li>Murphy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

Los procedimientos experimentales para el uso de animales de laboratorio fueron aprobados por el Cuidado de Animales institucional y el empleo de la Universidad de California, San Francisco (UCSF).

Presentamos aquí un método para la fundición de cerebrovasculature ratón adulto que puede ser fotografiada con diversas modalidades. La aplicación de este método a la BAVM modelo de la enfermedad permite el análisis en 3D de los vasos irregulares. Potencial de los futuros estudios incluyen la cuantificación de micro-CT imágenes, el análisis histológico de los tejidos, y el diagnóstico de la progresión de la enfermedad vascular. Problemas comunes y Sugerencias Perfusión completa de la vasculatura del cerebro no siempre se logra. Tener la presión suficiente para llegar a los vasos sanguíneos distal sin romper la aurícula izquierda o en la aorta es un reto. Para superar esto, aplique una presión constante de aproximadamente 100 mmHg. Además, ajuste de posición de aguja para asegurar flujo adecuado de fundición de agente en la aorta. Es importante que las arterias carótidas no se limitan por lo que el agente de casting puede llegar a los vasos cerebrales. Los procedimientos fundamentales que se relacionan con la mejora pequeña embarcación porde fusión son: (1) limpieza adecuada de la sangre de los vasos, (2) la filtración de la agente de casting, y (3) colocación de la aguja durante la inyección de agente de casting. Se debe tener cuidado para evitar la inyección de agente de fundición en la aurícula izquierda y el pulmón. Otros investigadores han logrado perfundidos ratón de 3 y 4 cerebrovasculature rata sin utilizar procedimientos adicionales. Análisis manual de los vasos perfundidos Microfil contrastados con hematoxilina mostró 93% del lumen de perfusión vaso 5. Siempre deseche o completamente limpia cualquier herramienta que se pone en contacto con el agente de casting, ya que se polimeriza rápidamente, y las herramientas no se puede utilizar para varios temas. Aclaración del tejido del cerebro del ratón rodea puede ser un reto. Después de la inmersión en salicilato de metilo, el tejido puede aparecer opaco, lo que hace difícil imágenes embarcación. Garantizar la correcta deshidratación y aclaración debe resolver este problema. La colocación de tejido cerebral en una mecedora en alcohol metílico y el salicylate tratamiento puede mejorar la clarificación de tejidos. Inconvenientes de la técnica Las limitaciones de esta técnica son: (1) es necesario para inyectar la solución de agente de casting inmediatamente después de mezclarlo, ya que se espesa rápidamente después de que el catalizador se agrega, (2) el agente de casting llena de grandes vasos de conductancia con facilidad, pero no fiable llenar recipientes más pequeños, tales como las arteriolas (vasos de resistencia) y capilares (vasos nutritivos), y (3) no es el mejor contraste radio-opaco para las micro-TC, aunque algunos grupos prefieren Microfil de micro-TC 1,6. Sin embargo, a raíz de las sugerencias propuestas por encima, incluso colocación de la aguja, la posición del buque y la presión de perfusión, la fundición completa de los buques más pequeños se puede lograr. La adaptabilidad a las diferentes herramientas de análisis que hace este agente en una herramienta valiosa para el análisis de la estructura cerebral, incluyendo la circulación arterial y venosa, con variosperspectivas. Aplicaciones de la técnica La ventaja de este método es su potencial de aplicación general. El reparto se llena todos los buques, incluyendo las arterias, venas y capilares de todos los tejidos dentro del cuerpo, por lo que las estructuras vasculares en múltiples órganos de diferentes especies pueden ser analizados. Anteriormente, otros grupos han usado esta perfusión de grandes 7 y pequeños 8 buques en humanos, el mono 9, 10 ovejas, ratas 4,11,12, y el ratón 3,13. Vamos a demostrar la evidencia de la utilización de esta técnica en el análisis de la estructura de la microvasculatura del cerebro del ratón, en los estados sanos y enfermos 2. Además, hay soluciones Microfil con viscosidades disponibles para perfundir los vasos de diferentes tamaños y diferentes colores para mejorar la visualización de los vasos en varios órganos. Por ejemplo, el rojo puede verse fácilmente en el parénquima cerebral pálido y amarillo contrasta conde color rojo oscuro miocardio. Por otra parte, ya que el material es radiopaco, vasos se pueden crear imágenes con micro-tomografía computarizada para obtener una imagen en 3D rotativo. El tejido también se puede utilizar para cortes de parafina y el análisis histológico después de ser fotografiado. Conclusión Estamos aquí demostrar un protocolo para hacer un reparto vascular de los vasos sanguíneos del cerebro del ratón adulto, que puede ser adaptado con una variedad de técnicas de análisis para estudiar la estructura morfológica de la cerebrovasculature. También hemos proporcionado evidencia de la aplicación de este método a un modelo de estado de la enfermedad BAVM, representado por los buques ampliada, de forma irregular que la derivación de sangre arterial a la circulación venosa. Hay una variedad de métodos de fundición disponibles vascular. El protocolo simple que ofrecemos aquí un elenco vascular puede ser utilizado como una herramienta para examinar el sistema vascular del cerebro del ratón adulto utiliza diversas modalidades de imagen.

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> Número de catálogo </td><td> Notas </td></tr><tr><td> Microfil </td><td> Flowtech, Inc. </td><td> MV130 </td><td> 4 meses de vida útil </td></tr><tr><td> Salicilato de metilo </td><td> Fisher Scientific </td><td> O3695-500 </td><td> para aclarar el tejido </td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Vascular imagen es crucial en el diagnóstico clínico y el tratamiento de las enfermedades cerebrovasculares, tales como las malformaciones arteriovenosas cerebrales (BAVMs). Los modelos animales son necesarios para el estudio de la etiopatogenia y el potencial de las terapias de las enfermedades cerebrovasculares. Imágenes de la vasculatura en animales grandes es relativamente fácil. Sin embargo, el desarrollo de métodos de imagen buque de modelos murinos de enfermedades cerebrales es deseable debido al coste y la disponibilidad de líneas de ratones modificados genéticamente. Imágenes de la murino árbol vascular cerebral es un reto. En los seres humanos y animales de mayor tamaño, el estándar de oro para evaluar la angioarquitectura en el macrovascular (conductancia) es el nivel de rayos X del catéter de contraste basado en la angiografía, un método no es adecuado para pequeños roedores. En este artículo, presentamos un método de fundición cerebrovascular que produce un esqueleto resistente del lecho vascular completo, incluyendo las arterias, venas y capilares que pueden ser analizados usando muchos dmodalidades ifferent. Fundición completa de los microvasos de la cerebrovasculature ratón puede ser difícil, sin embargo, estos desafíos se abordan en este protocolo paso a paso. A través de la perfusión intracardíaca del material de fundición vascular, todos los vasos del cuerpo están fundidas. Entonces, el cerebro se puede quitar y aclarado con el salicilato de metilo orgánico solvente. Imágenes tridimensionales de los vasos sanguíneos del cerebro se puede visualizar sencilla y económica con cualquier microscopio de campo claro convencional o un microscopio de disección. El cerebrovasculature fundido también se pueden visualizar y cuantificar el uso de micro-tomografía computarizada (micro-CT) 1. Además, después de haber sido fotografiado, el cerebro fundido puede ser embebido en parafina para su análisis histológico. La ventaja de este método de fundición vascular en comparación con otras técnicas es su amplia adaptación a diversas herramientas de análisis, incluyendo el análisis de campo claro microscópica, la TC debido a las características radiopacaspropias de la materia, así como el análisis histológico e inmunohistoquímico. Este uso eficiente de los tejidos puede salvar el uso de los animales y reducir los costos. Recientemente hemos demostrado la aplicación de este método para visualizar los vasos sanguíneos irregulares en un modelo murino de BAVM adultos a nivel microscópico 2, y ofrecer más imágenes de los vasos malformados fotografiada por micro-TAC. Aunque este método tiene sus inconvenientes y no puede ser ideal para todo tipo de análisis, es una técnica sencilla, práctica que puede ser fácilmente aprendido y aplicado ampliamente a la fundición vascular de los vasos sanguíneos en todo el cuerpo.

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

En este artículo presentamos una técnica sencilla y práctica para la fundición cerebrovascular que es fácil de realizar y pueden ser utilizados para obtener imágenes del árbol vascular del cerebro del ratón adulto.

Fundición cerebrovascular del ratón adulto de imágenes en 3D y el análisis morfológico

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

Research

JoVE Journal

Neuroscience

21.4K vistas.  University of California, San Francisco. En este artículo presentamos una técnica sencilla y práctica para la fundición cerebrovascular que es fácil de realizar y pueden ser utilizados para obtener imágenes del árbol vascular del cerebro del ratón adulto.

Video: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Investigación

Neurociencias

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Time-lapse de imágenes de Migración neuroblasto en rodajas agudas del cerebro anterior ratón adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Los métodos para la modulación y Análisis de NF-KB dependiente de la neurogénesis adulta

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

Patrones morfológicos potenciales evocados visuales corticales específicos del estímulo

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Evaluación morfológica y funcional de sinapsis de cinta en regiones de frecuencia específica de la Cóclea de Ratones

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Angiografía por fluorescencia para la evaluación de la perfusión del aneurisma y la paciencia de la arteria parental en modelos de aneurisma de rata y conejo

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análisis cinemático 3D para la evaluación funcional en el modelo de rata de lesión de aplastamiento de nervios ciáticos

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Preparación de la superficie coclear en el ratón adulto

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imágenes y análisis del transporte de neurofilamento en el nervio tibial de ratón extirpado

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...