Esta investigación fue apoyada por conceder R01NS062842 del Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares, Institutos Nacionales de Salud (VVD) y por subvenciones R21 NS064403 del Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares, Institutos Nacionales de Salud a través de ARRA (VVD) y R21 Instituto Nacional EB006301 de Imágenes Biomédicas y Bioingeniería, Institutos Nacionales de Salud (VVD).

El método es adecuado para, embebidos en parafina secciones de tejido fijadas con formaldehído. La técnica se demuestra aquí en su aplicación para el accidente cerebrovascular experimental en rata. 1. Componentes Preparación para tiramida amplificación de señal (TSA) Sistema Aunque la mejora basada en la TSA se utiliza en la etapa final de etiquetado, la preparación para esta reacción puede tomar más de 1 hora. Por lo tanto es conveniente para hacer los reactivos TSA antes de iniciar el etiquetado. <ol><li> Hacer tiramida fluoróforo solución madre. Utilice el reactivo tiramida fluoróforo en viales individuales con el equipo de marcaje de la TSA. Añadir 300 l de DMSO al vial de reactivo tiramida. Solución Fluoróforo tiramida valores es estable durante al menos 3 meses cuando se almacenan a 4 ° C. </li><li> Hacer tampón de lavado que contiene TNT: 0,1 M de Tris-HCl, pH 7,5; 0,15 M de NaCl; 0,05% de Tween-20. Si es necesario 0,3% de Triton X-100 puede ser sustituido por el 0,05% de Tween-20. AlternatIvely PBS puede ser utilizado como tampón de lavado. </li><li> Hacer TNB tampón de bloqueo que contenía: 0,1 M Tris-HCl, pH 7,5; 0,15 M de NaCl; 0,5% reactivo de bloqueo (suministrada con el kit). Para disolver completamente el reactivo de bloqueo, se añade lentamente en pequeños incrementos para el buffer mientras se agita. Calentar esta solución gradualmente a 55 ° C con agitación continua. Esto puede tomar 30 a 60 min. La solución aparecerá lechoso. Llevar a temperatura ambiente antes de usar. Alícuota y almacenar a -20 ° C para su uso a largo plazo. </li></ol> 2. Preparación de Secciones Utilice diapositivas montadas con 5 secciones micras de espesor cortadas a partir de tejidos fijados con formalina. <ol><li> Tratar secciones con xileno durante 15 min. </li><li> Rehidratar las siguientes secciones: remojar en 96% EtOH 2x durante 5 minutos cada uno; remojo en 80% EtOH durante 5 min; lavar con agua 2 veces durante 5 minutos cada uno. </li><li> Hacer la solución de trabajo proteinasa K mediante la dilución de la proteinasa K solución madre a 50 g / ml en PBS. </li><li> Añadir proteinasaK solución de trabajo a las secciones. Incubar 15 minutos a 23 º C. Pueden necesitar ser ajustado en función del tamaño de las secciones y tipos de tejidos El volumen de la solución y el tiempo de digestión. Para una sección pequeña (por debajo de 5 mm de diámetro) 50 l de solución de proteinasa serían suficientes. Sección Para un tamaño medio (~ 10 mm de diámetro), añadir 100 l de solución de proteinasa K. Por muy grandes secciones del volumen de la solución que se puede expandir. En todos los casos, evite que overdigestion de secciones que conduce a la pérdida completa de la señal y destruye la morfología del tejido. Esto sucede a menudo si el tiempo de incubación supera los 25 min. </li><li> Lávese las secciones de 2x agua destilada durante 5 minutos cada uno. Preparar la mezcla de etiquetado que contiene la sonda activa, mientras que las secciones están en el agua. </li></ol> 3. La tinción basada en TOPO VACC <ol><li> Prepare la solución de la sonda activa mediante la combinación de los siguientes: Agua - 11.75 l 1 M de Tris-HCl, pH 7.4 a 1.25 μl 100 pmol de 12-base de suicidio voladizo oligonucleótido - 1 l 100 pmol de oligonucleótido adaptador - 1 l 50 pmol VACC TOPO - 10 l </li><li> Mezclar suavemente con la pipeta. Incubar a temperatura ambiente (23 ° C) durante 15 min para permitir la activación de la sonda. </li><li> Tome secciones del tarro de lavado y aspirar el agua restante. A continuación, aplicar la solución de la sonda activo preparado en el paso 3.1 (25 l por ~ sección 10 x 10 mm). </li><li> Cubreobjetos las secciones y se incuba durante 15 minutos a 23 º C. Dip se desliza en el agua para eliminar suavemente cubreobjetos. Lave en agua 3 veces durante 5 minutos cada uno. </li></ol> 4. TSA de señal de mejora <ol><li> Cubrir las secciones de tejido con tampón de bloqueo TNB preparado anteriormente e incubar los portaobjetos en una cámara humidificada durante 30 min a temperatura ambiente (23 ° C). </li><li> Preparar una dilución 1:100 de conjugados de peroxidasa de rábano picante estreptavidina (SA-HRP) en TNB tampón de bloqueo. Aspirar TNBtampón de bloqueo y aplique la solución SA-HRP. Incubar los portaobjetos con SA-HRP durante 30 min a temperatura ambiente. Utilizar volumen de reactivo adecuado para cubrir la sección de tejido, generalmente de 100 l por sección. </li><li> Lavar los portaobjetos 3x por cada 5 min en tampón de lavado TNT (o PBS) a temperatura ambiente. </li><li> Prepare una dilución 1:50 de fluoróforo tiramida stock en 1x Amplificación Diluyente del kit. Pipetear la solución de trabajo tiramida fluoróforo en cada diapositiva. Utilice solución de trabajo suficiente para cubrir completamente la sección de tejido, generalmente de 100 l por sección. Haga solución de trabajo tiramida fluoróforo inmediatamente antes de cada etiqueta. La solución no puede ser reutilizado, por lo que descarta cualquier porción no utilizada de la misma después de la marcación. </li><li> Incubar los portaobjetos a temperatura ambiente durante 3 a 10 min. Monitorear incremento de fluorescencia con un microscopio hasta que los primeros indicios de estructuras se pueden ver. Proceder rápidamente a lavarse paso. </li><li> Lavar los portaobjetos 3x por cada 5 min en tampón de lavado TNT (o PBS) en sala de temperatura con agitación. </li><li> Añadir la solución de antifading con DAPI y cubreobjetos. Observar bajo el microscopio de fluorescencia. Para visualizar DAPI y FITC fluorescencia utilizan un conjunto de filtros de paso de banda: D490/40 excitación FITC, emisión de 520/10; DAPI D360/40 de excitación, emisión 460/20, o similar. </li></ol>

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	<li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J Leukoc Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., M&#252;ller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp Neurol. 196 (2), 290-297 (2005).</li><li>Faustino, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+contribute+to+endogenous+brain+defenses+after+acute+neonatal+focal+stroke.">Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke.</a> J Neurosci. 31 (36), 12992-13001 (2011).</li><li>Thored, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+accumulation+of+microglia+with+proneurogenic+phenotype+concomitant+with+persistent+neurogenesis+in+adult+subventricular+zone+after+stroke.">Long term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke.</a> Glia. 57 (8), 835-849 (2009).</li><li>Sierra, A., Abiega, O., Shahraz, A., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus-faced+microglia:+beneficial+and+detrimental+consequences+of+microglial+phagocytosis.">Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis.</a> Front Cell Neurosci. 7 (6), (2013).</li><li>Samejima, K., Earnshaw, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trashing+the+genome:+Role+of+nucleases+during+apoptosis.">Trashing the genome: Role of nucleases during apoptosis.</a> Nat.Rev. Mol. Cell Biol. 6 (9), 677-688 (2005).</li><li>Minchew, C. L., Didenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+detecting+the+phagocytic+phase+of+apoptosis:+enzyme-substrate+complexes+of+topoisomerase+and+DNA.">Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA.</a> Molecules. 16 (6), 4599-4614 (2011).</li><li>Shuman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+classes+of+DNA+end-joining+reactions+catalyzed+by+vaccinia+topoisomerase.">Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase.</a> Sci. Rep. 267, 16755-16758 (1992).</li><li>Barcia, C., 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=ROCK/Cdc42-mediated+microglial+motility+and+gliapse+formation+lead+to+phagocytosis+of+degenerating+dopaminergic+neurons+in+vivo.">ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo.</a> Sci. Rep. . 2, 809 (2012).</li><li>Didenko, V. V., Ngo, H., Minchew, C. L., Boudreaux, D. J., Widmayer, M. A., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+irreparable+ischemic+damage+in+brain+by+selective+labeling+of+double-strand+blunt-ended+DNA+breaks.">Visualization of irreparable ischemic damage in brain by selective labeling of double-strand blunt-ended DNA breaks.</a> Mol. Med. 8, 818-823 (2002).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

En este video se demuestra, en el formato de la sección de tejido, como etiquetar los fagocitos activos que clara la muerte celular en la isquemia cerebral focal. La técnica presentada es el primer enfoque que marca específicamente sólo las células de gestión de residuos, que han afectado y digerir activamente cadáveres celulares. Esto hace que sea ventajoso con respecto a otros métodos existentes para el etiquetado de las células fagocíticas, que no tienen esta capacidad. El méto...

El accidente cerebrovascular isquémico induce cambios profundos en el tejido cerebral afectado. Se inicia la muerte celular masiva, que conduce a la rápida activación de las células fagocíticas residentes de origen microglial. También pone en marcha la infiltración de cerebro isquémico por varios tipos de fagocitos profesionales derivadas de la sangre incluyendo neutrófilos, macrófagos, dendríticas, y mastocitos 1,2. Todavía se debate si esta respuesta a la lesión isquémica desempeña un positivo o un papel negativo. Aunque la fagocitosis después del accidente cerebrovascular puede ser beneficioso, ya que borra las células muertas y suprime la inflamación, sino que también genera especies reactivas de oxígeno tóxicos que afectan la supervivencia neuronal y exacerbando el daño tisular 1-5. Mientras que varios tipos de células fagocíticas infiltran cerebral isquémico, no todos ellos participan en la gestión de residuos por que envuelve los cuerpos celulares y despejando el camino para los procesos regenerativos 1-3. Este creates un requisito para la identificación selectiva de las células de gestión de residuos que llevan a cabo aclaramiento fagocítica de muerte celular en el accidente cerebrovascular. Cuando proyección de imagen tales células de gestión de residuos activos, también es importante responder a la cuestión de la eficiencia con que se degradan los cadáveres celulares envueltos. La degradación de la efectiva y completa del ADN de la célula que muere en la fagocitosis es esencial, ya que impide la auto-inmunización y la liberación de material nuclear patológica 6. Aquí presentamos nuevas sondas que utilizan saltos de ADN específicas como marcadores de células fagocitarias activas. Estas interrupciones de la firma se producen exclusivamente durante la ruptura de núcleos envueltos dentro de las células de gestión de residuos funcionales. Por lo tanto las sondas marcadoras selectivamente sólo los fagocitos que engullen y digieren activamente cadáveres celulares. También permiten la observación de la intensidad y la finalización de la ruptura de ADN después de la inmersión. Las sondas son útiles en la evaluación de la intensidad y efficirencia del despacho fagocítica. Las nuevas sondas se basan en un marcador no basado en proteína-y por lo tanto pueden ser particularmente ventajosa en los estudios de accidente cerebrovascular, donde extensa daño isquémico puede alterar la morfología celular o agotar marcadores a base de proteínas, especialmente dentro de la zona isquémica núcleo. El principio de la técnica se presenta en la Figura 1. La figura muestra las sondas de oligonucleótidos en forma de horquilla-ligados por la enzima topoisomerasa de vaccinia (VACC TOPO) al ADN 5&#39;OH termina generada por lisosomal ADNasa II durante la digestión de ADN 7.

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			<td height="34" style="height:34px;width:312px;">Name of the reagent or material</td>
			<td style="width:99px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:129px;">Comments (optional)</td>
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			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Xylene</td>
			<td>JT Baker</td>
			<td>9490-22</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Buffer</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaccinia Topoisomerase I</td>
			<td colspan="2">Vivid Technologies</td>
			<td>5 pmol/&#956;L stock&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-Base Overhang Suicide Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3&#8217; ; B &#8211; Biotin-dT; 100 pmol/&#956;L stock in ddH2O&#160;&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Adapter Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-TAG AAT CGT ATC-3&#8217;; 100 pmol/&#956;L stock in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M Tris-HCl, PH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">TSA, Fluorescein System&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>NEL701A001KT</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coverslips</td>
			<td></td>
			<td></td>
			<td style="width:129px;">22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Rat Brain Sections</td>
			<td></td>
			<td></td>
			<td style="width:129px;">Rat brain taken 48 hours after reproduction of ischemic stroke as described in 11. 5-6&#956;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Video camera and software</td>
			<td>Micromax</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td>Metamorph</td>
			<td></td>
			<td></td>
		</tr>
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assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

Se describe un nuevo método histoquímico para la visualización del despacho fagocítica en la isquemia cerebral focal. El enfoque permite el estudio de la eliminación de las células muertas en el accidente cerebrovascular por los fagocitos de gestión de residuos de cualquier linaje celular. Aunque numerosas células de diferentes orígenes que son capaces de fagocitosis están presentes en el cerebro isquémico, sólo una parte de ellos engullir activamente y digerir cadáveres de células. La visualización selectiva, cuantificación y análisis de dicha gestión de residuos activa fagocítica son útiles en la evaluación de la respuesta cerebral a la isquemia. Despacho eficiente la muerte celular es importante para la recuperación de la lesión isquémica cerebral, ya que abre el camino para que los procesos de regeneración posteriores. La falta de limpieza de los cadáveres daría lugar a una reacción tóxica causada por el ADN no degradado y proteínas. El procedimiento descrito utiliza sondas fluorescentes ligadas selectivamente por una topoisomerasa viral a roturas en el ADN característicos producidos en todos los fagocitos durante la inmersióny la digestión de las células irreversiblemente dañado por la isquemia. El método es una nueva herramienta para la investigación de la reacción del cerebro a la lesión isquémica.

El principio de la técnica para el etiquetado de los fagocitos de gestión de residuos activos se presenta en la Figura 1. El esquema demuestra que el producto de detección en tres pasos. La primera etapa comprende la activación de la sonda (Figura 1A); en el segundo paso la sonda activado se ligó a roturas en el ADN específicas en las secciones de tejido (Figura 1C); la tercera etapa incluye la amplificación de la señal fluorescente (Figura 1D)....

Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Se presenta una nueva técnica de fluorescencia para selectivo etiquetado in situ de las células fagocitarias activas, que claro off cadáveres de células en el accidente cerebrovascular. El enfoque es importante para evaluar la reacción del cerebro a la isquemia debido a que sólo una pequeña proporción de los fagocitos presentes en el cerebro isquémico participar en el aclaramiento de la muerte celular.

La evaluación de Liquidación fagocítica de muerte celular en el accidente cerebrovascular experimental por ligables Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

assessing-phagocytic-clearance-cell-death-experimental-stroke

Research

JoVE Journal

Medicine

8.1K vistas.  Baylor College of Medicine.  Se presenta una nueva técnica de fluorescencia para selectivo etiquetado in situ de las células fagocitarias activas, que claro off cadáveres de células en el accidente cerebrovascular. El enfoque es importante para evaluar la reacción del cerebro a la isquemia debido a que sólo una pequeña proporción de los fagocitos presentes en el cerebro isquémico participar en el aclaramiento de la muerte celular. 

Video: Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Trasplante de células madre en un In vitro Simulación de isquemia / reperfusión modelo

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ensayo in vitro Liquidación mesoteliales que modela los pasos iniciales de la metástasis del cáncer ovárico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Carriage Experimental neumocócica Humanos

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

La muerte celular asociada con Mitosis anormal observada por Imagen confocal en vivo las células cancerosas

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Trasplante de células madre en Experimental contuso Modelo de Lesión de la Médula Espinal

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Experimental glaucoma inducido por inyección ocular de microesferas magnéticas

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Ligadura del conducto biliar en los ratones: Inducción de la injuria hepática inflamatoria y fibrosis por colestasis obstructiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa de Bengala Photothrombosis por imagen óptica confocal<em&gt; En Vivo</em&gt;: Un modelo de solo recipiente Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...