Este trabajo fue apoyado en parte por subvenciones de los Institutos Nacionales de Salud (P30 HL101302 y RO1 HL109439 a JC) y la American Heart Association (SDG 5270005 a JC). A. Barazia fue apoyado por una beca de formación T32HL007829 NIH.

1. Preparación de microscopio intravital (Figura 1A) <ol><li> Preparar tampón superfusión (125 mM NaCl, 4,5 mM KCl, 2,5 mM CaCl 2, 1 mM MgCl 2, y 17 mM NaHCO 3, pH 7,4). </li><li> Girar en un baño de agua circulatorio para mantener la temperatura de tampón y una manta termo controlada a 37 ° C. Airear tampón con gas nitrógeno (5% de CO 2 en equilibrio con nitrógeno). </li><li> Encienda el sistema de microscopio (Sutter Lambda DG-4 de alta velocidad longitud de onda cambiador, estación de trabajo, Olympus microscopio BX61W, MPC-200 multi-manipulador, el ROE-200 controlador de fase, cámara de alta velocidad y multiplicador). </li><li> Para el modelo de inflamación vascular, utilizar un espejo dicroico 100%. Para inducir la formación de trombos por lesión con láser, sustituir un espejo 100% con un espejo de 50/50% y activar un sistema de ablación láser micropoint. </li></ol> 2. Preparación del músculo cremáster para microscopía intravital (Figura 1B) <ol><li> Anestesiar a unratón macho (6-8 semanas de edad, C57BL / 6) por inyección ip de ketamina (125 mg / kg de peso corporal (BW)) y xilazina (12,5 mg / kg de peso corporal). La Universidad de Illinois Cuidado de Animales institucional y el empleo Comisión aprobó todo cuidado de los animales y los procedimientos experimentales. Para un modelo de inflamación vascular, inyectar murino TNF-α (0,5 g en 250 l de solución salina) en un ratón intrascrotally 3 h antes de la formación de imágenes (2-2.5 horas antes de la cirugía). </li><li> Coloque el ratón sobre una manta térmica controlada a 37 º C en una bandeja de microscopio intravital. </li><li> Tire hacia arriba de la piel en la superficie del cuello utilizando fórceps y una incisión en la línea media horizontal de la piel del cuello hasta 1 cm con unas tijeras. </li><li> Abra suavemente el músculo cervical utilizando tijeras de punta roma y eliminar el músculo que rodea la tráquea. </li><li> Canular un tubo en la tráquea PE90 para eliminar la dificultad para respirar. </li><li> Retire suavemente el lado izquierdo del músculo cervical y aislar la vena yugular de los tejidos circundantes. </li><li> C annutarde un tubo PE10 en la vena yugular izquierda para la infusión de anticuerpos y anestésicos adicionales. </li><li> Tire suavemente hacia fuera y una incisión en la piel del escroto horizontalmente. Para mantener la integridad de los tejidos durante todo el experimento, tampón precalentado se superfused sobre el campo quirúrgico. </li><li> Pulse en la parte inferior del abdomen con una pinza y tire con cuidado un testículo con el fórceps otro. </li><li> Quite los tejidos conectivos que rodean todo el testículo. </li><li> Incisión en el músculo cremáster verticalmente y aplanar el músculo sobre un cubreobjetos de vidrio sobre una bandeja de microscopio intravital fijando la periferia. </li><li> Permitir 10-20 min para el músculo para estabilizar antes de la recogida de datos. Arteriolas del músculo cremáster (para la formación de trombos) y vénulas (para la inflamación vascular) con un diámetro de 30-45 micras fueron elegidos para el estudio. </li><li> Para mantener las condiciones de anestesia, infusión de 30-50 ml de la misma anestesia aproximadamente cada 30 minutos a través de la cannulus yugular. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 3. Microscopía intravital para TNF-α inducida por la inflamación vascular <ol><li> Después de la colocación del ratón en el microscopio intravital, abierto SlideBook software 5.0 y haga clic en &quot;Ventana de enfoque&quot; en la barra de menú para configurar la óptica adecuadas y objetivas (60X). </li><li> Infundir DyLight 488-rata conjugado anti-ratón CD42c (0,1 mg / g de peso corporal en 100 l de solución salina) y Alexa Fluor 647-conjugado anti-ratón de rata Gr-1 anticuerpos (0,05 mg / g de peso corporal en 100 l de solución salina) a través de un cannulus yugular . </li><li> Ir a la barra de menús y haga clic en &quot;Captura de imagen&quot; en la barra de herramientas. </li><li> Un nuevo menú aparecerá, en &quot;Imagen&quot;, seleccione &quot;Factor Bin&quot; como 1x1 y seleccione &quot;640&quot; en la anchura y &quot;460&quot; en la altura. </li><li> En &quot;Set Filter&quot;, marque la casilla de verificación de los canales que le gustaría exponer y definir el tiempo de exposición para cada uno. Por ejemplo, canal abierto: 10 mseg, FITC canal: 50 mseg, y Cy5 canal: 50 mseg. </li><li> Seleccione &quot;Time-Lapse Capture&quot; para ajustar el número de puntos de tiempo e intervalo. For el modelo de inflamación, el número de puntos de tiempo se establece en 1,000-1,500 con un intervalo de 200 ms (el tiempo total transcurrido: 5 min). </li><li> Haga clic en &quot;Test&quot; para ver una imagen de un solo avión en el tiempo de exposición especificado para ese canal. Ajuste el tiempo de exposición y / o la luz del microscopio hasta que el histograma muestra una distribución de intensidad suficiente (para captura multi-canal, repita este proceso para cada canal). </li><li> Una vez que todos los parámetros se ajustan (canales, tiempos de exposición, etc) en la &quot;Captura de imagen&quot;, seleccione &quot;Start&quot; para comenzar la captura. </li><li> Monitorear las plaquetas rodantes y adherentes y los neutrófilos durante 5 min en la mitad superior de una vénula cremáster inflamado en el modelo de inflamación vascular. De ocho a diez vénulas diferentes se registran en un ratón. </li><li> Las imágenes fueron tomadas con un aumento total de 60x (1,0 NA objetivo de inmersión en agua), dando un tamaño de ventana de 157 m x 118 micras. </li><li> Al finalizar el experimento, los ratones fueron sacrificados mediante cervical dislocation. </li></ol> 4. Análisis de datos para el modelo de inflamación vénulas <ol><li> Abra SlideBook software 5.0 y haga clic en &quot;carpeta&quot; en la barra de menú para abrir un archivo. </li><li> Haga clic en el menú de canales desplegable y seleccionar &quot;Abrir con FITC, Cy5, etc&quot; </li><li> Haga clic en &quot;renormalizar (barras de color rojo y verde)&quot; y seleccione el canal. Arrastre las barras rojas y verdes a la izquierda oa la derecha para optimizar o menos la señal de fluorescencia. </li><li> Haga clic en &quot;Aplicar&quot; y &quot;OK&quot; para actualizar la imagen. </li><li> Haga clic en botón &quot;Miniaturas&quot; para seleccionar la visualización actual como predeterminado. </li><li> Juega imagen capturada timelapse, y contar neutrófilos rodantes y adherentes durante el período de 5 min. </li><li> Para el análisis de la formación del trombo plaquetario, vaya a &quot;Máscara&quot; y seleccione &quot;Crear&quot;. </li><li> Nombre una máscara y la marca &quot;A imagen actual&quot;. </li><li> Haga clic en &quot;icono de lápiz grande&quot; en &quot;Marquee Tool&quot; en la vista principal. </li><li> Colorea una región fuera del recipiente en la vista principal para establecer una máscara de fondo. </li><li> Ir to &quot;máscara&quot;, haga clic en &quot;Copiar Esta Plane&quot;, haga clic en &quot;máscara Copia en punto de tiempo actual&quot; y seleccione &quot;Todos los puntos de tiempo&quot;, y haga clic en &quot;OK&quot;. </li><li> Para el cálculo de la señal de fondo, vaya a &quot;Estadísticas&quot; y haga clic en &quot;Estadísticas máscara&quot;. </li><li> Haga clic en &quot;Current Time Lapse 2D o imagen 4D (s)&quot; en el ámbito de imagen y seleccione &quot;Máscara entera&quot; en Ámbito Mask. En Funciones, seleccione &quot;Tiempo transcurrido (hh: mm: ms)&quot; en Fecha de captura, seleccione &quot;Area (píxeles)&quot; bajo Morfometría, seleccione &quot;Nivel máximo&quot; bajo Intensidad, y haga clic en &quot;Exportar&quot; (Este método estadístico nos permite calcular auto-fluorescencia intensidad (señal de fondo)). </li><li> Abrir el archivo de texto en MS Office Excel y calcular el valor medio de la señal de fondo durante todo el período de registro. Esta es la intensidad de fluorescencia de fondo. </li><li> Para determinar la intensidad de trombos, haga clic en &quot;icono de lápiz grande&quot; de nuevo en el &quot;Marquee Tool&quot; en la vista principal. </li><li> Color en el interior del recipiente en la vista principal para ajustar la señal de plaquetas. </li><li> Vaya a &quot;Mask&quot;, haga clic en &quot;Copiar Esta Plane&quot;, haga clic en &quot;máscara Copia en punto de tiempo actual&quot;, seleccione &quot;Todos los puntos de tiempo&quot;, y haga clic en &quot;OK&quot;. </li><li> Vaya a &quot;Estadísticas&quot; y haga clic en &quot;Estadísticas máscara&quot;. </li><li> Haga clic en &quot;Current Time Lapse 2D o imagen 4D (s)&quot; en el ámbito de imagen y seleccione &quot;Máscara entera&quot; en Ámbito Mask. En Funciones, seleccione &quot;Tiempo transcurrido (hh: mm: ms)&quot; en Fecha de captura, seleccione &quot;Area (píxeles)&quot; bajo Morfometría, seleccione &quot;Nivel Sum&quot; en intensidad, y haga clic en &quot;Exportar&quot; (Este método estadístico nos permite calcular la intensidad de fluorescencia en el interior del recipiente). </li><li> Abra el archivo de texto en Microsoft Office Excel. La señal de fluorescencia de las plaquetas se calcula restando el valor medio de la intensidad de área de fondo x (píxel) de la suma de la intensidad en el interior de la vasija. Esta es la intensidad de fluorescencia de los trombos de plaquetas. </li><li> Repita esto en 30 vénulas diferentes en 3-5 ratones diferentes. A continuación, calcular el valor de la mediana de la intensidad de fluorescencia de las plaquetas. </li><li> Aanalizar la rodadura de neutrófilos y la adherencia, la afluencia de rodadura de neutrófilos se determinó durante 5 min en cada vénula y presentados como número de células por minuto. El número de neutrófilos adherentes que eran estacionaria durante&gt; 30 seg y se arrastró lentamente (&lt;10 micras durante 30 s), pero no rodar, se contaron y se presentaron como el número de células por 5 min. </li></ol> 5. Microscopía intravital de trombosis inducida por láser arteriolar 5-1 de calibración de láser de ablación <ol><li> Colocar un portaobjetos de microscopio reflejado en el escenario, aplicar una pequeña gota de agua destilada a la parte superior de la corredera, y ajustar la intensidad y el enfoque utilizando el ocular. </li><li> Abra &quot;Ventana Focus&quot; y seleccione la opción &quot;FRAP&quot; en la pestaña SlideBook software 5.0. </li><li> Ajuste la potencia del láser en 40-50, y ajustar &quot;repeticiones doble clic&quot; y del &quot;tamaño doble clic&quot; a 8. Marque en la &quot;guía&quot; casilla para mostrar un conjunto de punto de mira. </li><li> Seleccione la opción &quot;Arrow&quot; icono de la barra de tareas on la parte superior de la pantalla. </li><li> En la sección &quot;Alineación FRAP&quot; haga click en &quot;Fire siguiente&quot;. Un pequeño punto debería aparecer cerca de la esquina inferior izquierda de la pantalla. Una vez que la mancha aparece, haga doble clic en el centro de la misma. Una vez que se hace clic, otro punto debería aparecer por encima ya la derecha del punto primero, haga doble clic en él. Repita esto para 16 puntos (puntos 16 º aparecerá en la esquina inferior derecha.) Haga clic en &quot;Guardar&quot; cuando haya terminado de actualizar los parámetros de calibración. </li><li> Para probar la exactitud de la calibración, haga clic en el &quot;Centro&quot; botón, un pequeño punto debe aparecer en el centro de la pantalla. Si no es así, repita la calibración hasta que la precisión deseada. </li></ol> 5-2 inducida por láser formación de trombos arteriolar <ol><li> Coloque un ratón anestesiado (véase el procedimiento de 2-2 a 2-12) en la platina del microscopio. </li><li> En virtud de la &quot;captura&quot; de la ventana, seleccione un protocolo o crear uno nuevo. Los ajustes son los siguientes: CY5 (70 ms de exposición), Open (10 mseexposición c), y FITC (50 exposición mseg). La imagen se capturó más de 20 minutos (aproximadamente 2.400 puntos de tiempo con un intervalo de tiempo de 500 ms). </li><li> Infundir DyLight 488-anti-ratón conjugado CD42c y Alexa Fluor 647-anti-ratón conjugados Gr-1 anticuerpos como se describe anteriormente. </li><li> Optimizar los parámetros de microscopio incluyendo la intensidad de fluorescencia y la imagen de campo brillante como se describe en 3-3 a 3-7. </li><li> Haga clic en el botón &quot;Test&quot; para asegurar la intensidad adecuada campo claro o señal de anticuerpos, la colocación de las arteriolas y enfoque. </li><li> Haga clic en &quot;Inicio&quot; para iniciar el proceso de captura de imágenes. </li><li> Antes de disparar el láser de ablación, asegúrese de que el icono del puntero del ratón ha sido seleccionado y que la pared del vaso está enfocada. </li><li> Para disparar el láser, haga doble clic en un punto de 2-3 micras interna de la pared del vaso. Lesión de las células endoteliales de las arteriolas provoca un cambio de forma visual que deben ser evidentes en la imagen de campo claro, seguido por la acumulación de plaquetas. Monitor de tromboformación de 5 min después de la lesión con láser. </li><li> Cinco minutos después de la lesión con láser (el tamaño del trombo ahora debe ser estable) pausa y cancelar la captura. Posteriormente, iniciar una captura con 2400 puntos de tiempo con un intervalo de 500 ms para registrar las plaquetas adherentes y neutrófilos rodantes / adherentes durante 20 min. De tres a cinco arteriolas son registrados en un ratón. </li><li> Al finalizar el experimento, los ratones fueron sacrificados mediante dislocación cervical. </li></ol> 6. Análisis de datos para el modelo de trombosis arteriolar <ol><li> Repase los pasos 4.1 a 4.5. </li><li> Para obtener la intensidad de la fluorescencia de fondo durante la formación de trombos de plaquetas, pasar a través de los pasos 4,7 4,14. </li><li> Vaya a &quot;Mask&quot;, haga clic en &quot;segmentos&quot;, seleccione FITC en el canal, e insertar el valor medio de la señal de fondo en Low. Haga clic en &quot;Aplicar&quot; y &quot;OK&quot;. </li><li> Para analizar la intensidad de fluorescencia de trombos de plaquetas (DyLight 488-conjugado anti-CD42c anticuerpo), </strong&gt; repasar los pasos 4,18 por 4,19. Para el cálculo de la señal de anticuerpos, &quot;Intensidad Sum&quot; se resta de la señal de fondo (&quot;Intensidad máxima&quot; x &quot;en la zona (píxeles)&quot;). Esta es la intensidad de fluorescencia de los trombos de plaquetas. </li><li> Para cuantificar los neutrófilos rodantes y adherentes, jugar el timelapse y contar el número de células que visiblemente rodar sobre el trombo plaquetario más de 20 min. Rodadura se define como una disminución en la velocidad de neutrófilos, mientras que la interacción con el trombo de plaquetas durante al menos 2 segundos. Neutrófilos adherentes se definen como cualquier neutrófilos que permanecen unidos a los trombos de plaquetas durante al menos 2 min. El número de neutrófilos rodantes y adherentes se presentó como el número de células por 20 min. La velocidad de rotación se calculó utilizando un sistema de rastreo de partículas. </li></ol>

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Los autores declaran no tener ningún interés en competencia financiera.

Aquí se describe un protocolo detallado para tiempo real microscopía de fluorescencia intravital para visualizar heterotípicas plaquetas neutrófilos interacciones en el endotelio activado durante la inflamación vascular y trombosis. Anteriormente, los enfoques similares microscópicas de fluorescencia se registraron para estudiar el mecanismo molecular de la formación de trombo y la inflamación vascular. 8,12 Desde el heterotípicos interacción célula-célula podría ser importante para la oclusión ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

La interacción de las plaquetas activadas y leucocitos (principalmente neutrófilos) en el endotelio activado trombosis media y la inflamación vascular. 1,2 Durante la formación de un trombo en el sitio de la lesión arteriolar, las plaquetas adherentes al endotelio activado y proteínas subendoteliales matriz de soporte de rodadura de neutrófilos y la adherencia. 3 A la inversa, bajo venulares condiciones inflamatorias, neutrófilos adherentes al endotelio activado puede soportar la adhesión y acumulación de plaquetas circulantes. Heterotípicos plaquetas agregación de neutrófilos requiere procesos secuenciales por las interacciones con los receptores específicos de receptor-contador entre las células. 4 Se sabe que las células endoteliales activadas liberan moléculas de adhesión tales como el factor de von Willebrand, iniciando así la adhesión de las plaquetas y la acumulación bajo condiciones de alto cizallamiento. 5 Además, células endoteliales activadas soporte rodante de neutrófilos y la adhesión por selectinas que expresan unmolécula-1 d adhesión intercelular (ICAM-1), respectivamente, en condiciones de bajo cizallamiento. 4 de plaquetas P-selectina interactúa con los neutrófilos a través de P-selectina glicoproteína ligando-1 (PSGL-1), induciendo de ese modo la activación de neutrófilos β2 integrinas y firmes adhesión entre dos tipos de células. A pesar de los avances en los experimentos in vitro en el que heterotípicas plaquetas neutrófilos interacciones se determinan en sangre entera o de células aisladas, 6,7 esos estudios no se pueden manipular las condiciones de estrés oxidativo durante la enfermedad vascular. En este informe, utilizando marcadas con fluorescencia, anticuerpos específicos contra un ratón de plaquetas y neutrófilos marcador, se describe un protocolo detallado intravital microscópico para monitorear las interacciones heterotípicas de plaquetas y neutrófilos sobre el endotelio activado durante TNF-α inducida por inflamación o por láser inducida lesión del músculo cremáster en microvasos de ratones vivos.

Usando un análisis detallado microscopía intravital, heterotípicas plaquetas neutrófilos interacciones en el endotelio activado se visualizaron mediante la infusión de anticuerpos marcadas con fluorescencia contra una de plaquetas (CD42c) o neutrófilos marcador (Gr-1) en ratones vivos. En un modelo de TNF-α inducida por inflamación venular, neutrófilos más de rodadura se adhirieron de forma estable al endotelio presumiblemente por la interacción de las integrinas β2 activados con...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Aquí se presenta una técnica experimental de microscopía intravital de fluorescencia para visualizar heterotípicas plaquetas neutrófilos interacciones en el endotelio activado durante la inflamación vascular y la formación de trombos en ratones vivos. Esta tecnología microscópica será valiosa para estudiar el mecanismo molecular de la enfermedad vascular y para poner a prueba los agentes farmacológicos bajo condiciones fisiopatológicas.

Imágenes en tiempo real de heterotypic plaquetas neutrófilos Interacciones en el endotelio activado durante la inflamación vascular y la formación de trombos en ratones vivos

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K vistas.  University of Illinois at Chicago. Aquí se presenta una técnica experimental de microscopía intravital de fluorescencia para visualizar heterotípicas plaquetas neutrófilos interacciones en el endotelio activado durante la inflamación vascular y la formación de trombos en ratones vivos. Esta tecnología microscópica será valiosa para estudiar el mecanismo molecular de la enfermedad vascular y para poner a prueba los agentes farmacológicos bajo condiciones fisiopatológicas.

Video: Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Imágenes en tiempo real de los leucotrienos B 4 La migración mediada por células y BLT1 interacciones con β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Investigación

Inmunología e Infección

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Imágenes de la adhesión de leucocitos al endotelio vascular en la presión intraluminal de alta

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

La detección de microARNs en la microglía por PCR en tiempo real en el SNC normal y durante la neuroinflamación

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

En tiempo real de imágenes en vivo de las células T de señalización Formación de complejos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imágenes en tiempo real de la célula-célula cruces endoteliales Durante neutrófilos Transmigración Bajo Flujo Fisiológica

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

Una Novela<em&gt; In vitro</em&gt; Modelo para el estudio de las interacciones entre Whole Sangre humana y del endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Descifrar e imagen Patogénesis y Cording de<em&gt; Abscessus Mycobacterium</em&gt; En embriones de pez cebra

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrófilos y monocitos en mesentérica venas por Microscopia intravital en ratones anestesiados en Tiempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Caracterización metabólica de polarizado M1 y macrófagos derivados de Médula ósea M2 Uso del análisis extracelular Flux en tiempo real

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...