Esta investigación fue apoyada por el INSERM, la Universidad de Aix-Marseille y un premio de Desarrollo Profesional de HFSPO obtenida por CL Forestier.

Declaración de Ética: se realizaron todos los estudios en animales, de acuerdo con las directrices y protocolos que fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Aix-Marsella, Francia. Mujer C57Bl / 6 ratones a 8-10 semanas de edad se obtuvieron comercialmente y manejarse de acuerdo con las reglas del Decreto N ° 8 87-848, 19 de octubre de 1987, París. Todos los experimentos utilizando L. parásitos donovani LD1S se llevaron a cabo de acuerdo con normas de bioseguridad de la legislación francesa y la Unión Europea. 1. Ratón La infección con L. donovani Promastigote parásitos <ol><li> Preparar el medio de cultivo para el mantenimiento in vitro de L. parásitos promastigotes donovani, LD1S (MHOM / SD / 62 / 1S-CL2D). <ol><li> Utilice medio M199. Suplemento medio M199 con 10% de suero de ternera fetal (inactivado con calor a 56 ° C durante 30 min), 25 mM HEPES pH 6,9, 12 mM NaHCO 3, glu 1 mMtamine, RPMI 16040 mezcla de vitaminas, ácido fólico 10 mM, adenosina 100 mM, hemina 7,6 mM, 50 U / ml de penicilina y 50 mg / ml de estreptomicina. </li></ol></li><li> Cultura LD1S parásitos en 10 ml de medio completo M199 a 26 ° C en un matraz de 25 cm 2 ventilado. </li><li> El uso de sucesivas etapas de centrifugación diferencial, preparar una población de parásitos promastigotes que se enriquecen en formas metacíclicos. <ol><li> Cosechar un cultivo de parásitos mediados de la fase de registro. Cuente con un hemocytomer. Resuspender 5 x 10 5 parásitos / ml en 10 ml de medio completo M199. Mantener el cultivo del parásito en condiciones anaeróbicas hasta que alcanzan la fase estacionaria tardía (en aproximadamente 5-6 días). </li><li> Girar la cultura LD1S a 1200 xg durante 10 min a 26 ° C para sedimentar los parásitos no metacíclicos, recuperar el sobrenadante y girar de nuevo a 2500 xg a 26 ° C durante 10 min. Nota: Este sedimento final es altamente enriquecido en los parásitos promastigotes metacíclicos. </li><li> Resuspender el precipitado en 100 l de PBS. Retirar una pequeña alícuota para la fijación con 25% de glutaraldehído centésimas v: v y contar parásitos utilizando un hemocitómetro. </li></ol></li><li> Diluir la población de parásitos enriquecido con metacyclic a 1 x 10 7/100 l en PBS. Inyectar la suspensión parásito en la vena de la cola de un ratón anestesiado (ver a continuación) utilizando una jeringa de 1 ml con una aguja de 30 G. Para los animales de control, inyecte 100 l de PBS en la vena de la cola. </li></ol> 2. Procedimientos Quirúrgicos <ol><li> Desinfectar el espacio de trabajo con un spray desinfectante biocida y todos los instrumentos quirúrgicos con etanol al 70% durante 30 min. </li><li> Preparar una solución anestésica, de acuerdo con el peso del animal, que contiene 125 mg / kg de ketamina y 12,5 mg / kg de xilazina diluido en PBS. </li><li> Pesar el ratón y por vía intraperitoneal inyectar la dosis apropiada de anestesia utilizando una jeringa de 1 ml con una aguja de 30 G. </li><li> Mantenga el ratón cálido y compruebe que el ratón es completamente anestheTized pellizcando la almohadilla de la pata antes de proceder. Vuelva a inyectar una dosis media de la solución anestésica en el ratón cada 60 min. </li><li> Afeitarse el abdomen del ratón y limpiarlo con Vetedine. Hacer una pequeña incisión en la piel y la musculatura debajo de la caja torácica en el lado izquierdo del abdomen. </li><li> Coloque un pedazo de gasa húmeda sobre el abdomen justo por debajo de la zona abierta. Exponer el hígado y colocarlo en la gasa. Coloque otro pedazo de gasa húmeda sobre el hígado. </li><li> Colocar un 24 x 60 mm 2 a 150 micras de espesor cubreobjetos, cianoacrilato-unido a un armazón de metal, sobre el hígado para la visualización bajo un microscopio. </li><li> Proteja los ojos del ratón con una gasa húmeda. </li><li> Después de la sesión de imágenes, la eutanasia del animal anestesiado por dislocación cervical. </li></ol> 3. Microscopia intravital de imágenes de la arquitectura hepática <ol><li> Llevar a cabo los experimentos de microscopía intravital en un microscopio confocal invertido equipado con alcámara, un objetivo de inmersión 63X glicerol aceite apocromático (NA 1.4), argón (488 nm) y HeNe (543 nm, 633 nm) láseres y un diodo azul (405 nm) hermostatic controlada. </li><li> Coloca el ratón sobre la platina del microscopio con el cubreobjetos cubriendo la cara de hígado sobre el objetivo. Ajuste la temperatura de la cámara a 29 ° C. Ajuste el enfoque utilizando la autofluorescencia hígado para permitir la visualización de los sinusoides. </li><li> Para visualizar la vasculatura, preparar una solución de 500 mg BSA-Alexa 647 diluido en 100 l de PBS, para cada ratón. Inyectar esta solución por vía intravenosa en la vena de la cola del ratón anestesiado utilizando una jeringa de 1 ml con una aguja de 30 G. </li><li> Para visualizar los núcleos de las células hepáticas, preparar una solución de Hoechst 33342 en PBS. Inyectar esta solución a 8 mg / kg de ratón por vía intraperitoneal usando una jeringa de 1 ml con una aguja de 30 G. </li><li> Utilice el modo secuencial normal, el objetivo de inmersión 63X y el escáner en 400 Hz a la imagen de la hepáticanúcleos de las células y la vasculatura hepática. Encienda el 405 nm diodo azul para Hoechst excitación y el láser de 633 nm para BSA-Alexa 647 de excitación. Encienda el láser de argón 488 nm y definir una banda adquisición grande para visualizar el autofluorescencia hígado. </li></ol> 4. Microscopia intravital de imágenes del hígado para la medición de flujo sanguíneo velocidad <ol><li> Abra el software LAS del microscopio (LAS-AF visor Versión 3.1.0 build 8587). Seleccione el modo de escáner de resonancia al abrir el software de microscopio. </li><li> Haga clic en la pestaña de configuración para configurar el hardware. Haga clic en láser y seleccione el láser HeNe 633. Haga clic en configuración y seleccione una resolución de 12 bits. </li><li> Haga clic en el menú de adquisición. En la ventana de configuración de la vía de haz, seleccione el objetivo 63X y establecer la potencia del láser 633 a 100%. Active la ganancia de la señal y fuera de los parámetros establecidos por la selección de Alexa-633 en el menú desplegable PM1-3. </li><li> En la pestaña de adquirir, seleccione la adquisición &#39;XYT&#39;de modo. Ancho formato establecido en 1024 x 1024. Seleccione la velocidad a 8.000 Hz. Seleccione el agujero de 3 unidades de Airy. Seleccione un factor de zoom de 3. No seleccione el modo bidireccional. </li></ol> 5. Análisis Cuantitativo de RBC Velocity Utilizando los XYT Imágenes (Método 1) <ol><li> Haga clic en el icono de &#39;Live&#39; para visualizar el flujo sanguíneo en un modo directo. Seleccione el área de interés y seleccione un vaso sanguíneo específico para el análisis. Ajuste el vaso de interés a la posición horizontal mediante el ajuste de la rotación de campo de coordenadas de escaneo de &quot;X&quot;, &quot;Y&quot; y en el panel de control USB. </li><li> Detenga el modo &#39;Live&#39; una vez que el vaso de interés se fija. Cambie el conjunto de ancho formato a 1024 x 256 en el modo de adquisición de la ventana, la línea media = 1, marco Media = 1 ajuste, seleccione &quot;pilas&quot; 150. Haga clic en &#39;inicio&#39; para adquirir imágenes. </li><li> Para el análisis cuantitativo de la velocidad de RBC usando las imágenes &quot;XYT&quot; abren el software ImageJ. Seleccione la opción &#39;Archivo&#39;pestaña en ImageJ, haga clic en &quot;Abrir&quot; y luego elegir el archivo de su interés. </li><li> Vaya al menú &quot;plugin&quot; en ImageJ, seleccione Opciones LOCI y Bio-formatos de importación. En esta ventana, seleccione la pila de visualización parámetros como &#39;HyperStack&#39;. En la opción de orden de apilamiento, seleccione la opción: &quot;xyzct&quot;. Nota: Se abrirá una ventana con toda la serie de adquisiciones. </li><li> Haga clic en &quot;Plugins&quot; en el menú y seleccione la opción ImageJ MTrackJ. Haga clic en el icono &#39;Añadir&#39; en la pequeña ventana y haga clic en el RBC para realizar un seguimiento. Haga clic en la misma RBC en cada trama siguiente y haga clic en el icono de &quot;medida&quot; para medir la velocidad. Nota: El archivo generado se pueden guardar en formato .exl. </li><li> El seguimiento de un mínimo de cinco glóbulos rojos por buque y analizar un mínimo de tres vasos individuales del mismo tamaño. </li></ol> 6. Análisis Cuantitativo de RBC velocidad utilizando la línea xt Imágenes (Método 2) <ol><li> Haga clic en &#39;Live&#39; para visualizar el f sangrebaja en el modo directo. Seleccionar un vaso sanguíneo específico para el análisis. Ajuste el vaso de interés a la posición horizontal. Detenga el modo &#39;Live&#39; una vez que el vaso de interés se fija. </li><li> Seleccione el modo de escaneo &quot;xt&quot; y establecer el lumen central del buque seleccionado a lo largo de la línea de exploración. Conjunto ancho formato en 1024 x 512, velocidad = 8,000Hz, línea media = 32, tiempo = 512. Haga clic en &quot;Inicio&quot; y adquirir las imágenes de líneas xt. </li><li> Generar rayas para el análisis cuantitativo de la velocidad de RBC abriendo el archivo .lif y la apertura de la imagen de la línea xt de interés. En el software LAS-AF, seleccione &quot;experimentos&quot;, abra el .lif y seleccione la imagen. Nota: se abrirá automáticamente una quimógrafo. </li><li> Medir el tiempo (t, obtenido en el eje Y) requerida para una racha de partículas (que muestra la reflexión oscuro) para recorrer una cierta distancia (d, obtenido en el eje X) en este quimógrafo. Seleccione la herramienta &quot;dibujar la línea&quot; y trazar una línea horizontal a la racha. Tenga en cuenta la distancia enmicras y el tiempo en segundos que se generan en la pestaña resultado en la parte inferior de la imagen. </li><li> Calcula la velocidad como V = distancia / tiempo usando los valores obtenidos a partir de la quimógrafo. </li><li> Cuantificar un mínimo de cinco partículas rayas imagen por xt y un mínimo de tres vasos individuales del mismo tamaño. </li></ol>

<ol>
	<li>Vollmar, B., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hepatic+microcirculation:+mechanistic+contributions+and+therapeutic+targets+in+liver+injury+and+repair.">The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair.</a> Physiol. Rev. 89, 1269-1339 (2009).</li><li>Srinivasan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+blood+flow+measured+by+optical+methods.">Absolute blood flow measured by optical methods.</a> SPIE Newsroom. , (2011).</li><li>Seifalian, A. M., Stansby, G. P., Hobbs, K. E., Hawkes, D. J., Colchester, A. C. <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+liver+blood+flow:+a+review.">Measurement of liver blood flow: a review.</a> HPB. Surg. 4, 171-186 (1991).</li><li>Engwerda, C. R., Ato, M., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages,+pathology+and+parasite+persistence+in+experimental+visceral+leishmaniasis.">Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis.</a> Trends Parasitol. 20, 524-530 (2004).</li><li>Beattie, L., 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=Dynamic+imaging+of+experimental+Leishmania+donovani-induced+hepatic+granulomas+detects+Kupffer+cell-restricted+antigen+presentation+to+antigen-specific+CD8+T+cells.">Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.</a> PLoS Pathog. 6, e1000805 (2010).</li><li>Lee, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intravascular+immune+response+to+Borrelia+burgdorferi+involves+Kupffer+cells+and+iNKT+cells.">An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.</a> Nat. Immunol. 11, 295-302 (2010).</li><li>Geissmann, F., 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=Intravascular+immune+surveillance+by+CXCR6++NKT+cells+patrolling+liver+sinusoids.">Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.</a> PLoS Biol. 3, e113 (2005).</li><li>Marques, P. E., 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=Imaging+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat. protocols. 10, 258-268 (2015).</li><li>Thiberge, S., 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=In+vivo+imaging+of+malaria+parasites+in+the+murine+liver.">In vivo imaging of malaria parasites in the murine liver.</a> Nat. protocols. 2, 1811-1818 (2007).</li><li>Vacchina, P., Morales, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+screening+test+using+Leishmania+promastigotes+stably+expressing+mCherry+protein.">In vitro screening test using Leishmania promastigotes stably expressing mCherry protein.</a> Antimicrob. Agents Chemother. 58, 1825-1828 (2014).</li><li>Ferrer, M., Martin-Jaular, L., Calvo, M., del Portillo, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+spleen:+quantitative+analysis+of+parasite+mobility+and+blood.">Intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood.</a> J. Vis. Exp. , (2012).</li><li>Kamoun, W. S., 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=Simultaneous+measurement+of+RBC+velocity,+flux,+hematocrit+and+shear+rate+in+vascular+networks.">Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks.</a> Nat. Methods. 7, 655-660 (2010).</li><li>MacPhee, P. J., Schmidt, E. E., Groom, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittence+of+blood+flow+in+liver+sinusoids,+studied+by+high-resolution+in+vivo+microscopy.">Intermittence of blood flow in liver sinusoids, studied by high-resolution in vivo microscopy.</a> Am. J. Phys. 269, G692-G698 (1995).</li></ol>

The authors have nothing to disclose.

El reciente desarrollo de la microscopía intravital del hígado del ratón abre nuevas posibilidades para la investigación de la respuesta fisiológica a la infección in vivo y en tiempo real 5,9,10. El flujo sanguíneo de órganos es un parámetro fisiológico crítico que a menudo se altera en muchas enfermedades. Sin embargo, el estado de la hemodinámica hepática en condiciones fisiológicas e infecciosas sigue siendo un área poco explorada. En este estudio, los métodos basados ​​en IVM...

Hemodinámica órgano-específicas son importantes características fisiológicas de cualquier órgano de mamífero. Anomalías en el flujo sanguíneo puede ser la consecuencia de la inflamación y un signo de disfunción de órganos 1. Por lo tanto, el flujo de sangre organización, estructura y función aparecen como parámetros críticos para el análisis en condiciones fisiológicas y patológicas. Las técnicas que se han utilizado comúnmente para el análisis de flujo de sangre en un órgano específico contienen varias limitaciones, incluyendo el límite de resolución de la técnica en sí (por ejemplo, formación de imágenes Doppler del flujo sanguíneo), la capacidad para la medición del flujo sanguíneo absoluta solamente (volumen de sangre por unidad que sirve un órgano) (por ejemplo, la tomografía de coherencia óptica) y la medición de los cambios en la velocidad promedio en una población grande y heterogéneo de los vasos sanguíneos 2,3. El sistema circulatorio del hígado vincula diferentes subtipos de los vasos que son heterogéneos en su tamaño, estructura y función. Enesta tecnología de imagen estudio, microscopía intravital (IVM) se aplica para evaluar la hemodinámica del hígado in vivo, en tiempo real, en alta resolución y en paralelo para descubrir las características de los vasos sanguíneos individuales que componen el órgano hepático. El reciente desarrollo de esta poderosa técnica de imagen óptica permite al investigador para recolectar datos dinámicos sobre los animales que viven en una alta resolución espacial y temporal. Al permitir la visualización directa y el monitoreo en tiempo real de los procesos biológicos específicos y rápidos en vivo, IVM ofrece una oportunidad única para el investigador a los vasos sanguíneos imagen individual, y medir y cuantificar la velocidad de los glóbulos rojos individuales (RBC) dentro de un seleccionado específicamente buque hepática. En este estudio, hemos implementado la técnica de IVM en el hígado del ratón para investigar la influencia de la infección del ratón por el parásito Leishmania hepatotropos en la hemodinámica hepática. L. donovanies el agente responsable de la leishmaniasis visceral, una enfermedad grave caracterizada por respuestas inflamatorias agudas-sobre-crónica y una patología que está presente en múltiples órganos, incluyendo el bazo y el hígado. En un modelo experimental de ratón de leishmaniasis visceral, la infección del hígado es auto-resolver mientras que la infección esplénica es progresiva 4. Estos resultados de la infección por Leishmania con respecto a los órganos individuales son todavía no completamente entendidos. Investigación de la hemodinámica de hígado y bazo en condiciones patológicas arrojará nueva luz sobre las interacciones huésped-parásito y patogénesis de la enfermedad. Nuestro sistema modelo experimental se basa en la exposición y la obtención de imágenes del hígado de un ratón anestesiado que recibió la inyección intravenosa de tintes fluorescentes específicas para el etiquetado de la intravasculature hepática. El hígado es un órgano favorable para microscopía intra-vital. Después de realizar un pequeño incision en el abdomen, el hígado se exterioriza y se coloca en una gasa húmeda, luego en un cubreobjetos con el objetivo de reducir los artefactos de movimiento debido a latidos del corazón y la respiración suavemente. El hígado se coloca entonces dentro de la vista de una lente de microscopio. En comparación con el nodo linfático y bazo que requiere el uso de dos fotones para los estudios de microscopía de IVM, la ventaja de que el hígado se encuentra en su arquitectura 3D homogénea / anatomía que permite el uso de un microscopio confocal convencional, con una profundidad máxima de penetración de aproximadamente 50 micras, para obtener imágenes de microscopía intravital 5-8. Este estudio describe dos métodos de imagen independientes para la medición cuantitativa de RBC velocidad y la velocidad de flujo sanguíneo en los vasos sanguíneos individuales. En el primer método, el flujo de sangre del hígado se adquiere usando un modo de bi-dimensional xy con el tiempo. Los datos XYT resultantes se analizan usando el plugin MtrackJ en el software ImageJ libre, que permite el seguimiento de individUAL glóbulos rojos en el tiempo. En el segundo método, se selecciona un solo vaso sanguíneo y su correspondiente flujo de sangre se analiza usando el análisis de la línea modo de adquisición rápida del microscopio de escaneo láser confocal. El vaso de interés se escanea a alta frecuencia a lo largo de su eje central a través de una línea axial. La velocidad del flujo sanguíneo se cuantifica a continuación, en base a la diferencia de contraste entre los eritrocitos y el plasma oscuros sin etiqueta marcada con fluorescencia. Las intensidades de fluorescencia de los glóbulos rojos y el plasma adquiridos a lo largo de la línea de exploración se representan gráficamente contra el tiempo para obtener rayas, los ángulos de las cuales son proporcionales a las velocidades de un RBC individual. El objetivo de este artículo es proporcionar un método simple y reproducible para la imagen y la medición de la velocidad del flujo sanguíneo dentro de los vasos sanguíneos individuales del hígado y de poner a disposición las herramientas básicas para el buen desarrollo de la cirugía ratón, IVM y análisis cuantitativos de la velocidad glóbulos rojos individuales. Tsu enfoque permitirá a los investigadores a obtener nuevos conocimientos sobre la velocidad de la sangre en condiciones patológicas.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Alexa 647</td>
			<td>lifetechnologies</td>
			<td>A34785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC 500 mol wt</td>
			<td>SIGMA</td>
			<td>46947</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>PanPharma</td>
			<td>20434</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>KP07KEU</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetedine</td>
			<td>Pharma Animal</td>
			<td>6869029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate liquid</td>
			<td>Cyanolit</td>
			<td>5833300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip frame: Membrane slide for microdissection part N&#176;: 5013</td>
			<td>Molecular machines</td>
			<td>50103</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip DiaPath 24x60 ep: 1.6 mm</td>
			<td>DiaPath</td>
			<td>61061</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica&#160;</td>
			<td>TCS-SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-AF viewer&#160;</td>
			<td>Leica software</td>
			<td>Version 3.1.0 buid 8587</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

Microscopía intravital (IVM) es una poderosa técnica de imagen óptica que ha hecho posible la visualización, monitoreo y cuantificación de diversos eventos biológicos en tiempo real y en los animales vivos. Esta tecnología ha avanzado enormemente nuestra comprensión de los procesos fisiológicos y fenómenos patógenos mediada en órganos específicos. En este estudio, IVM se aplica al hígado de ratón y protocolos están diseñados para imagen in vivo el sistema circulatorio del hígado y medir los glóbulos rojos (RBC) de velocidad en vasos hepáticos individuales. Para visualizar los diferentes subtipos de los vasos que caracterizan el órgano hepático y llevar a cabo mediciones de la velocidad del flujo sanguíneo, C57BL / 6 ratones se inyectaron por vía intravenosa con un reactivo de plasma fluorescente que etiqueta la vasculatura hepática asociada. IVM permite in vivo, en tiempo real, la medición de la velocidad de RBC en un recipiente específico de interés. El establecimiento de esta metodología permitirá ainvestigar la hemodinámica hepática bajo condiciones fisiológicas y patológicas. En última instancia, esta metodología basada en la proyección de imagen será importante para el estudio de la influencia de L. infección donovani en la hemodinámica hepática. Este método se puede aplicar a otros modelos infecciosas y órganos de ratón y podría extenderse aún más para pre-clínica de pruebas del efecto de un fármaco sobre la inflamación mediante la cuantificación de su efecto sobre el flujo sanguíneo.

La organización arquitectónica específica de los sinusoides del hígado puede ser visualizado basa en la propiedad autofluorescente de este órgano (Figura 1, panel B y C, verde), la inyección intraperitoneal de Hoechst para el etiquetado de los núcleos de los hepatocitos (Figura 1B, azul) y la inyección intravenosa de BSA fluorescente para la tinción del sistema circulatorio hepática (Figura 1C, rojo). El hígado se compone de varios subtipos diferentes de los ...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Microscopia intravital de imágenes del hígado después<em&gt; Leishmania</em&gt; Infección: Una Evaluación de Hemodinámica hepática

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

intravital-microscopy-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.4K vistas.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Video: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Imágenes in vivo de los cultivos transgénicos parásitos Leishmania en un host en vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Investigación

Inmunología e Infección

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravital de los ganglios linfáticos inguinales

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagen intravital del timo de ratón utilizando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital del bazo: Análisis cuantitativo de la movilidad del parásito y el flujo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagen intravital del nodo linfático poplíteo ratón

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Muy resuelto Microscopía intravital rayas-iluminación de centros germinales

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un ensayo de adhesión de flujo para estudiar Leucocyte Reclutamiento para hepático humano sinusoidales endotelio en condiciones de estrés de cizalla

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Largo Plazo intravital Multifotónica de imágenes de microscopía de células inmunitarias en sanos y enfermos de hígado utilizando ratones CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Dos fotones Intravital proyección de imagen de leucocitos durante la respuesta inmunitaria en hígado de ratón tratados con lipopolisacárido

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Proyección de imagen de interacción de células en Mucosa traqueal durante la infección del Virus de la Influenza usando microscopia Intravital de dos fotones

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...