The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTA: El siguiente protocolo se adhiere a las directrices establecidas por la Junta de Revisión Institucional (IRB) de Georgia Tech. 1. Extracción de sangre y la Sangre Procedimiento de aislamiento celular de color rojo <ol><li> Recoger 40-120 ml de sangre de donantes en 10 ml tubos vacutainer heparinizados. Iniciar PBMC (células mononucleadas de sangre periférica) de aislamiento dentro de las 4 horas de la recolección. Durante este tiempo, mantener la sangre a temperatura ambiente. NOTA: O / N de almacenamiento de la sangre en RT o en 4 ° C no se recomienda ya que esto resultará en un menor rendimiento de PBMC. </li><li> Diluir la sangre completa 1: 1 en PBS estéril enfriado con hielo (4 ° C). </li><li> Pipetear 10 ml de helado polisacárido hidrofílico en un tubo vacío, estéril cónico de 50 ml. Transferir suavemente la sangre entera diluida en la parte superior del polisacárido hidrófilo. </li><li> Usar una pipeta de 25 ml en el ajuste lento y la sangre de la pipeta a lo largo del lado del tubo muy lentamente. Asegúrese de que la sangre no se mezcla con el sacárido antes de la centrifugation porque el polisacárido hidrófilo es tóxico para las células y de otra manera, el rendimiento PBMC será menor. </li><li> Centrifugar las muestras de sangre durante 30 minutos a 400 xg a 4 ° C. Si está disponible, reducir la velocidad de freno de la centrífuga a la mitad de la máxima para una mejor separación después de la centrifugación. </li><li> Aspirar la fracción de plasma / plaquetas de la muestra de sangre centrifugada. Aspirar con cuidado la capa de polisacárido hidrófilo directamente encima de la capa de glóbulos rojos envasados. </li><li> Recoger 8-10 ml de glóbulos rojos empaquetados y diluir 1: 1 con no estéril 0,9% NaCl solución salina. </li></ol> 2. Microscopía Confocal Evaluación de glucosilada Estructuras Clot (simulados diabéticos coágulos) <ol><li> Descongele fibrinógeno humano y la etiqueta fluorescente conjugado fibrinógeno humano a 37 ° C. </li><li> Pipetear lo siguiente en un 0,5 ml tubo de microcentrífuga se graduaron. <ol><li> Para los coágulos de fibrinógeno, normales sanos, mezclar fibrinógeno humano con 10% de fibrinógeno humano marcado con fluorescenciaconjugado en un tampón de Tris 50 mM / NaCl 100 mM a una concentración de 5 mg / ml. </li><li> Para fibrinógeno glucosilada artificialmente (para simular la formación de coágulos diabéticos), incubar fibrinógeno humano y 10% de fibrinógeno marcado con fluorescencia conjugado en una solución 5 mM de glucosa disuelta en 50 mM Tris / NaCl 100 mM para una concentración resultante de 6,8 mg / ml. </li></ol></li><li> Cubra los tubos de microcentrífuga utilizando un material opaco para evitar la exposición a la luz. Incubar los tubos en un 37 ° C baño de agua durante 48 hr. </li><li> Haga una cámara en un portaobjetos de microscopio de vidrio mediante la colocación de un adhesivo delgada en los 2 lados de la diapositiva. </li><li> Después del período de incubación de 48 horas, preparar reactivos para la formación de fibrina por la descongelación FXIIIa y trombina alfa humano a RT. </li><li> Diluir FXIIIa a 80 U / ml en tampón 5 mM CaCl 2. </li><li> Diluir la trombina a 4 U / ml en tampón 5 mM CaCl 2. <ol><li> Para coágulos normales, asegúrese de que las concentraciones finales de fibrinógeno, FXIIIa, y trombina son 2,5 mg / ml, 20 U / ml, y 1 U / ml, respectivamente, en un volumen de 50 l. </li><li> Para los coágulos glucosilada, asegúrese de que las concentraciones finales de fibrinógeno, FXIIIa, y la trombina son 3,4 mg / ml, 20 U / ml, y 1 U / ml, respectivamente, en un volumen de 50 l. </li></ol></li><li> Inmediatamente pipetear 30 l de la solución de fibrina en la cámara formada por el adhesivo. </li><li> Coloque un 22 mm x 22 mm cubreobjetos de vidrio con un espesor de 0,15 mm en la parte superior de las 2 capas de adhesivo. Selle los lados abiertos con adhesivo transparente para evitar que el coágulo de fibrina se sequen. Asegúrese de que el adhesivo no interactúa con el coágulo de fibrina. </li><li> Permita que los coágulos de fibrina que polimerizan a 21-23 ° C durante 2 horas antes de la imagen confocal. </li><li> Tomar imágenes de microscopía confocal de los coágulos utilizando un microscopio confocal con una lente de 40X / 1.30 Aceite de M27 con un láser de argón de 488 nm. </li></ol> 3. Microscopía Confocal Evaluación de fibrina coágulos que contengan hematíes falciformes pacientes de celulares <ol><li> Descongele fibrinógeno humano y la etiqueta fluorescente conjugado fibrinógeno humano a 37 ° C. </li><li> Pipetear lo siguiente en un 0,5 ml tubo de microcentrífuga se graduaron. <ol><li> Para el, coágulo de fibrina normal y saludable, la mezcla de fibrinógeno humano con 10% de conjugado marcado con fluorescencia fibrinógeno humano en un tampón de Tris 50 mM / NaCl 100 mM a una concentración de 5 mg / ml. </li><li> Para los coágulos que contienen SCD glóbulos rojos, mezclar fibrinógeno humano y 10% de conjugado marcado con fluorescencia fibrinógeno humano en un Tris 50 mM / NaCl 100 mM tal que la concentración resultante del fibrinógeno es 10 mg / ml. </li></ol></li><li> Etiquetar los glóbulos rojos aislados (tanto normales como los de pacientes con anemia drepanocítica obtenidos del protocolo de aislamiento RBC) utilizando una solución de marcaje celular. <ol><li> Suspender las células a una densidad de 1 x 10 6 por ml en cualquier medio de cultivo libre de suero elegido. </li><li> Añadir 5 l / ml de la suspensión celular a la solución de etiquetado celular. Mezclar bien con la pipeta suave suavemente. </li><li&gt; incubar durante 20 minutos a 37 ° C. </li><li> Centrifugar los tubos de suspensión etiquetados a 1.500 rpm durante 5 min a 37 ° C. </li><li> Eliminar el sobrenadante y suavemente volver a suspender las células en 37 ° C medio. </li><li> Repita el procedimiento de lavado (3.3.4 y 3.3.5) dos veces más. </li></ol></li><li> Diluir FXIIIa a 80 U / ml en tampón 5 mM CaCl 2. </li><li> Diluir la trombina a 4 U / ml en tampón 5 mM CaCl 2. <ol><li> Para la formación de coágulos normales, asegúrese de que las concentraciones finales de fibrinógeno, FXIIIa, y la trombina son 2,5 mg / ml, 20 U / ml, y 1 U / ml, respectivamente, en un volumen de 50 l. </li><li> Para los coágulos que contienen SCD glóbulos rojos, asegúrese de que las concentraciones finales de fibrinógeno, FXIIIa, y la trombina son 5 mg / ml, 20 U / ml, y 1 U / ml, respectivamente, en un volumen de 50 l. </li></ol></li><li> Añadir 10 l de los glóbulos rojos marcados en la solución de fibrina. Pipeta 30 l de la fibrina con eritrocitos en el cristal de microscopio (consulte deslice preparación para glcoágulos ycated). </li><li> Deje que la fibrina polimerizar a 21-23 ° C durante 2 horas antes de la imagen confocal. </li><li> Tome imágenes confocal de los coágulos utilizando el microscopio confocal, y utilizar láseres de excitación con longitudes de onda de 488 nm y 633 nm para excitar la fibrina marcada con fluorescencia y glóbulos rojos. </li></ol> 4. Microscopía Confocal Evaluación de fibrinólisis Tarifas en Estructuras Clot glicada <ol><li> Repita el protocolo de microscopía confocal de coágulos glucosilada (Protocolo Paso 2) para la evaluación del índice fibrinólisis en coágulos glucosilada. </li><li> No selle los extremos de la cámara con adhesivo transparente. Permita que los coágulos de fibrina que se polimerizan durante 1,5 horas a 21-23 ° C en un recinto cerrado que contenga 250 ml de agua para prevenir la deshidratación. </li><li> Después de la polimerización, obtener imágenes de los coágulos utilizando el microscopio confocal. </li><li> Pipetear plasmina en el extremo abierto de la cámara y dispersar la plasmina a través del coágulo a través de la acción capilar. <ol><li> Paralos experimentos fibrinolisis normales y glucosilada con concentraciones normales, pipeta 25 l a 200 g / ml de plasmina en la apertura de la cámara. </li><li> Para los coágulos glucosilada con concentraciones reducidas, pipeta 25 l de 50 mg / ml en la apertura de la cámara. </li></ol></li><li> Utilice la función de series de tiempo (véase la Figura 1) en el software de microscopio confocal para capturar en tiempo real imágenes de vídeo de la plasmina lisar el coágulo. <ol><li> Haga clic en el modo de adquisición y luego seleccione &quot;Time Series&quot;. Seleccione el número de ciclos y el intervalo de tiempo de grabación. </li></ol></li></ol> 5. Calcular Fibrinólisis Precios <ol><li> Determinar la tasa de lisis mediante el establecimiento de un área (2500 m 2) y calcular el tiempo transcurrido requerido para disolver un área fija del coágulo. Calcular la tasa de lisis utilizando la siguiente ecuación (Figura 2): <img alt="Ecuación 1" fo:content-width="2in" src = &quot;/ files / ftp_upload / 52019 / 52019eq1.jpg&quot; /&gt; </li></ol> 6. Análisis estadístico de la fibrinólisis Precios <ol><li> Analizar los datos de lisis utilizando software de análisis estadístico. Realizar un ANOVA de una vía y una prueba post hoc de Tukey-Kramer 44,45. </li></ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+extravascular+and+intravascular+cells+to+fibrin+network+formation,+structure,+and+stability.">Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability.</a> Blood. 114, 4886-4896 (2009).</li><li>Curtis, D. J., 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=A+study+of+microstructural+templating+in+fibrin-thrombin+gel+networks+by+spectral+and+viscoelastic+analysis.">A study of microstructural templating in fibrin-thrombin gel networks by spectral and viscoelastic analysis.</a> Soft Matter. 9, 4883-4889 (2013).</li><li>Kim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+fibrin+network+structure+and+mechanical+properties:+an+experimental+and+computational+analysis.">Correlation between fibrin network structure and mechanical properties: an experimental and computational analysis.</a> Soft Matter. 7, 4983-4992 (2011).</li><li>Keegan, P. M., Surapaneni, S., Platt, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+disease+activates+peripheral+blood+mononuclear+cells+to+induce+cathepsins+k+and+v+activity+in+endothelial+cells.">Sickle cell disease activates peripheral blood mononuclear cells to induce cathepsins k and v activity in endothelial cells.</a> Anemia. 2012, 201781 (2012).</li><li>Allison, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+sickle-cell+haemoglobin.">Properties of sickle-cell haemoglobin.</a> Biochemical Journal. 65, 212 (1957).</li><li>Kuehl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of experiments : statistical principles of research design and analysis. , (2000).</li><li>Lindman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of variance in experimental designs. , (1992).</li><li>Pan, W., Galkin, O., Filobelo, L., Nagel, R. L., Vekilov, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastable+Mesoscopic+Clusters+in+Solutions+of+Sickle-Cell+Hemoglobin.">Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin.</a> Biophysical Journal. 92, 267-277 (2007).</li><li>Wootton, D. M., Popel, A. S., Alevriadou, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+the+dissolution+of+mural+fibrin+clots+by+tissue-type+plasminogen+activator.">An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.</a> Biotechnology and bioengineering. 77, 405-419 (2002).</li><li>Sazonova, I. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=127+Mathematic+model+of+fibrin+clot+lysis+by+plasmin.">127 Mathematic model of fibrin clot lysis by plasmin.</a> Fibrinolysis and Proteolysis. 12, 45 (1998).</li></ol>

The authors have nothing to disclose.

Para obtener datos significativos sobre la estructura de los mecanismos de coagulación en estados de enfermedad, es importante aislar los factores implicados en la coagulación para determinar los efectos de las proteínas y células en estas condiciones. Este protocolo fue desarrollado para los fines de la investigación de la estructura del coágulo de fibrina en los estados diabéticos y SCD in vitro. Es necesario entender los mecanismos implicados en la formación de fibrina y ...

La lesión de revestimiento endotelial de un vaso sanguíneo se repara a través de la respuesta hemostática, o la formación de un coágulo de sangre. Cuando la sangre penetra en la matriz extracelular, factores tisulares activar las plaquetas en el torrente sanguíneo que facilitan la iniciación de la cascada de la coagulación. El componente mecánico clave de este proceso de curación es la matriz de fibrina, compuesto de fibras de fibrina que son altamente elástico, y puede sostener grandes fuerzas 1-4. Muchos investigadores han estudiado la estructura de formación, y la función de la fibrina ampliamente en las últimas décadas 5-13. Los pacientes con enfermedades como la diabetes mellitus y de células falciformes tienen un mayor riesgo de desarrollar complicaciones trombóticas tales como infartos de miocardio, enfermedad isquémica del corazón y accidentes cerebrovasculares 14-19. Más de 2 millones de personas son diagnosticadas con diabetes mellitus cada año en los Estados Unidos. Hay dos tipos de diabetes: Tipo I, donde elcuerpo no puede producir cantidades adecuadas de insulina, y Tipo II, donde el cuerpo se vuelve resistente a la insulina. Entre los pacientes con diabetes, enfermedad cardiovascular (ECV) es la causa del 80% de la morbilidad y la mortalidad asociadas con la enfermedad 20,21. La enfermedad de células falciformes (ECF) es un trastorno genético de la sangre que afecta a más de 100.000 personas en los Estados Unidos 22. SCD es una enfermedad mutación puntual que hace que las células rojas de la sangre para convertirse en forma de media luna, lo que hace difícil para que las células pasan a través de la vasculatura arterial 23. Ambos de estos estados de enfermedad aumentan las probabilidades de desarrollar condiciones aterotrombóticos en el cuerpo. Una de las razones de esto es un resultado de la estructura de fibrina alterada y función en estados de enfermedad 14,24-26. En la diabetes y la enfermedad de células falciformes, existe hipercoagulabilidad y la actividad hipofibrinolisis que induce la aterotrombosis y la enfermedad cardiovascular (CVD) en comparación con pacientes sanos 17,27,28. Se sabe que hipofibrinolisis promueve la progresión de la aterosclerosis y engendra eventos isquémicos recurrentes para los pacientes con enfermedad de la arteria coronaria prematura 29. En el manuscrito, se determinó el papel de las propiedades físicas de fibrina en este entorno particular. Estructuras coágulo de fibrina en pacientes no enfermos se componen de fibras finas, poros más grandes, y en general menos densa 14,24. Se ha encontrado que el aumento de la porosidad y coágulos de fibrina menos densas en pacientes sanos para facilitar la fibrinólisis 16. En condiciones hipertrombótico tales como la enfermedad de células falciformes y diabética, hay un aumento en la producción de fibrinógeno, haciendo que la concentración de fibrinógeno para aumentar los niveles normales de 2,5 mg / ml en pacientes sanos 30-33. Los coágulos de fibrina formados en los pacientes diabéticos se han encontrado para ser menos poroso, más rígido, tener más puntos de ramificación, y más denso en comparación con sanos, no-diacientes diabéticos 14,24,33-35. La estructura de fibrina alterada es un resultado de los mecanismos de glicación que se producen en las proteínas implicadas en la formación del coágulo. No enzimática (irreversible) glicación se produce cuando las moléculas de glucosa se ​​unen a residuos de lisina en la molécula de fibrinógeno, que inhibe factor humano XIIIa (FXIIIa) a partir de glutamina y lisina residuos adecuadamente de reticulación 33,36,37. El análisis estructural de las redes de fibrina ha sido ampliamente estudiado recientemente. En particular, los investigadores han utilizado la microscopía electrónica y la reconstrucción 3D de las redes de fibrina 38, investigados cómo ambas células intravasculares (endoteliales) y extravasculares (fibroblastos y músculo liso) células afectan a la estructura de fibrina 39, viscoelástico utilizado y el análisis espectral para analizar estructuras de fibrina 40, y correlaciones desarrolladas entre la estructura de fibrina y las propiedades mecánicas utilizando experimental y computacional enfoques 41 </sup&gt;. El objetivo del presente estudio fue formular estructuras coágulo en condiciones trombosis celular diabética hoz y simulados y utilizar la microscopía confocal para el examen de la estructura y función de los coágulos en estados de enfermedad. Los coágulos de fibrina se forman a partir de fibrinógeno humano, trombina humana, y FXIIIa. Los coágulos se lisaron usando plasmina. Para simular las condiciones diabéticas, aumento de la concentración de fibrinógeno se incubó en solución de glucosa para inducir in vitro la glicación fibrinógeno. Para simular las condiciones de coagulación anemia de células falciformes, el aumento de las concentraciones de fibrinógeno se mezclaron con hematocrito células falciformes recogido de pacientes como se ha hecho anteriormente por nuestro grupo 42. Estos métodos se utilizaron para examinar la estructura y las funciones que participan en la formación del coágulo de fibrina y la fibrinolisis bajo condiciones de enfermedad, así como los mecanismos que inducen CVD. En base a la información actual sobre estas enfermedades, las estructuras coágulo de fibrina glicosilada eran más densas con menos de unnd poros más pequeños. Los coágulos de fibrina con las células rojas de la sangre de pacientes con anemia drepanocítica (GR) eran también más denso y se muestran agregación de los glóbulos rojos y las agrupaciones de fibrina aglomerados. Este es un fenómeno bien establecido que se ha determinado previamente 43. También se planteó la hipótesis de que la tasa de fibrinólisis sería significativamente menor en los coágulos de fibrina glucosilada con y sin reducción de plasmina en comparación con saludable, fibrina normal. Los resultados mostraron que para los coágulos de fibrina glucosilada, resultados significativamente diferentes tipos de lisis se observaron sólo bajo condiciones de reducida concentración de plasmina. Esta técnica experimental de la utilización de la microscopía confocal ofrece ventajas significativas sobre otros métodos de imagen, porque las células y las proteínas permanecen en su estado nativo, lo que permite la captura de vídeo en tiempo real de la actividad de coagulación. Este método de coagulación sintéticamente inductor es también más barato y más eficiente en el tiempo que la obtención de muestras de pacientes y filtrar individuoproteínas y enzimas. Además, mediante el uso de proteínas y enzimas separadas para sintetizar coágulos, los coágulos se estandarizaron de modo que no era la variabilidad entre las muestras como resultado de otras proteínas en el plasma.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">PBS</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ficoll-Paque (hydrophilic polysaccharide)</td>
			<td style="width:287px;">GE Healthcare</td>
			<td style="width:119px;">45-001-749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml heparinized vacutainer tubes</td>
			<td style="width:287px;">BD Biosciences</td>
			<td style="width:119px;">366643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Human Fibrinogen</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 human fibrinogen conjugate</td>
			<td style="width:287px;">Molecular Probes</td>
			<td style="width:119px;">F13191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL graduated microcentrifuge tube</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose powder</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">15023-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FXIIIa</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VIS Confocal Microscope</td>
			<td style="width:287px;">Zeiss LSM 510</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50 mM Tris</td>
			<td style="width:287px;">Lonza</td>
			<td style="width:119px;">S50-642</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:315px;">Calcium Chloride (CaCl2)</td>
			<td style="width:287px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiD cell-labeling solution</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">L7781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Plasmin</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Chloride (5 M NaCl)</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Statistical Modeling Software</td>
			<td>IBM</td>
			<td style="width:119px;">SPSS Statistics 22</td>
			<td></td>
		</tr>
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</table>

experimental and imaging techniques for examining fibrin clot structures in normal and diseased states

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

Microscopía Confocal Análisis de Estructuras Clot glucosilada fibrina Las imágenes de microscopía confocal de coágulos normales y glucosilada se presentan en la Figura 3. Análisis de microscopía confocal de los coágulos normales y glucosilada revela que los coágulos glucosilada son más densos con poros más pequeños que los coágulos normales con y sin la adición de FXIIIa durante la polimerización coágulo. En la Figura 3A y 3B,</strong...

Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Técnicas Experimentales y de imagen para examinar la estructura coágulo de fibrina en normales y enfermos Unidos

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

experimental-imaging-techniques-for-examining-fibrin-clot-structures

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Unit 1

Cardiovascular Drugs: Anticoagulants and Antianginal Agents

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11.3K vistas.  Georgia Institute of Technology & Emory University School of Medicine. In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Video: Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

El tratamiento de los defectos osteocondrales de articulación de la rodilla del conejo mediante la implantación de células madre mesenquimales alogénicas en coágulos de fibrina

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Investigación

Medicina

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Una tubería de multimodalidad 3D Image Integración y Planificación asistida por ordenador en la cirugía de la epilepsia

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

La visualización del parénquima vascular y la regeneración después del 70% hepatectomía parcial en ratones normales

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Un clavo intramedular de bloqueo para estandarizada fijación del fémur osteotomías para analizar normal y defectuoso Bone Healing en ratones

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Seguimiento In Vivo del Desarrollo del Edema y Patología Microvascular en un Modelo de Malaria Cerebral Experimental usando Imágenes de Resonancia Magnética

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualización Cuantitativa del Infiltrado de Leucocitos en un Modelo Murino de Miocarditis Fulminante por Microscopía de Hoja Ligera

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

En Vivo Análisis morfométrico de los nervios craneales de humanos usando resonancia magnética en la enfermedad orejas y oídos de la audiencia Normal de Menière

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

Resonancia Magnética Cardíaca para la Evaluación del Trombo Cardíaco Sospechoso: Técnicas Convencionales y Emergentes

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...