Esta investigación fue apoyada por el Programa de Investigación Intramural del Instituto Nacional de Artritis y Enfermedades Musculoesqueléticas y de la piel de los Institutos Nacionales de Salud.

1. Recolección de sangre entera <ol><li> Obtener un mínimo de 10 ml de sangre periférica de los individuos afectados, así como la edad y el sexo controles sanos. La sangre venosa se deberían recoger en tubos vacutainer con heparina sódica de recolección de sangre, en lugar de utilizar otros anticoagulantes como EDTA, y se invierte varias veces para asegurar una mezcla adecuada con la heparina sódica. Tenga en cuenta que 10 ml es el volumen mínimo de sangre requerido para la siembra en las muestras por duplicado para este ensayo en particular. </li><li> Las muestras deben ser procesadas tan pronto como sea posible después de la recolección para obtener mejores resultados. Sin embargo, la sangre heparinizada se pueden almacenar a temperatura ambiente bajo condiciones de poca iluminación para un máximo de 24 horas hasta que esté listo para ser procesado (2). </li></ol> 2. Preparación de sangre entera <ol><li> Centrifugar todas las muestras de sangre a 3.000 rpm (con freno desactivado) durante diez minutos a temperatura ambiente. Sin alterar la capa leucocitaria, el plasma se pueden obtener de la parte superior y se almacena para otros fines experimentales. Las muestras de plasma se puede almacenar temporalmente a -20 ° C o -80 ° C para el almacenamiento a largo plazo </li><li> Una vez que el plasma se recoge, lavado de las muestras por resuspensión de los glóbulos rojos (GR) de pellets y la capa leucocitaria con suero libre (incompleta) RPMI 1640 los medios de comunicación (a temperatura ambiente). Pipeta suavemente la mezcla en los tubos de recogida y traslado a un tubo cónico de 50 ml. </li><li> Volumen hasta 50 ml con los medios de comunicación incompleta de cultivo RPMI 1640 y suavemente invierta los tubos para crear una mezcla homogénea. Centrifugar las muestras a 3000 rpm (con freno desactivado) durante diez minutos. Asegurarse de que exista una separación clara, aspirar el sobrenadante, teniendo cuidado de no perturbar la capa leucocitaria. Repita este procedimiento 2-4 veces para asegurarse de que todos los contaminantes y las citocinas obligado se retiran antes de la estimulación. </li><li> Después del último lavado, resuspender el precipitado de glóbulos rojos y la capa leucocitaria de modo que el volumen total es de 20 ml (para 10 ml de sangre total extraída inicialmente) con incompleta RPMI 1640 los medios de comunicación. El volumen total se puede ajustar en función del volumen de sangre extraída inicialmente. </li><li> Uso de la Visión Cellometer, sangre diluida se puede contar sin glóbulos rojos lisis por la elección de la opción de glóbulos blancos cuentan con naranja de acridina (AO). Añadir una parte de la sangre diluida con AO una parte, y carga de 20 l de la mezcla de 1:1 en una cámara de recuento desechables. La concentración final de AO después de la dilución debe ser 1μg/mL. </li><li> Los ajustes necesarios con incompleta RPMI 1640 los medios de comunicación a una concentración celular final de 2.0 x 10 6 células / ml. </li></ol> 3. La estimulación de la sangre entera <ol><li> El ensayo requiere un mínimo de cuatro condiciones (cada uno hizo por duplicado): estimular, además de LPS solo, la adición de ATP sola, y finalmente LPS más ATP se suman. Añadir 1 ml del preparado 2.0 x 10 6 células / ml de sangre total diluida en el pozo apropiado de una placa de tejido de 24 pocillos. </li><li> Los estimulantes debe ser reconstituido y que alcance la temperatura ambiente antes de comenzar el ensayo de estimulación. Liofilizado LPS y el ATP debe ser reconstituido de acuerdo a las instrucciones del fabricante. Tras la reconstitución, LPS y el ATP puede ser diluida a una concentración de existencias adecuada utilizando incompleta RPMI 1640 los medios de comunicación. </li><li> Añadir a la LPS LPS sólo, así como a la LPS más ATP y por lo que la concentración final de trabajo de LPS es 1 mg / ml. La duración de la estimulación total es de 3 horas. Sin embargo, la sangre es estimuladas con LPS durante 2 horas y 40 minutos. Las muestras se colocarán en una incubadora a 37 ° C y 5% CO 2. </li><li> ATP se agrega durante los restantes 20 minutos del período de estimulación 3 horas. Añadir a la ATP ATP y solo el LPS y así ATP y las muestras de lugar en la incubadora. La concentración final de trabajo de ATP es de 2 mm. </li><li> Después del período de incubación de 3 horas, recoger los sobrenadantes por alícuotas de cada muestra en un tubo de microcentrífuga de 1,5 ml y posteriormente centrifugar las muestras a 10.000 rpm durante 2 minutos a temperatura ambiente. Transferir el sobrenadante a otro conjunto de tubos de 1,5 microcentrífuga. </li><li> Las muestras pueden analizarse inmediatamente o almacenada temporalmente a -20 ° C o -80 ° C para almacenamiento a largo plazo. </li></ol> 4. Citoquinas ensayo <ol><li> Las muestras que se congelan necesidad de equilibrar a temperatura ambiente antes del análisis. Las concentraciones de citocinas para IL-1βcan se evaluó a través de una variedad de métodos. Utilizamos el Bio-Plex citoquina pro humanos x compleja ensayo con el Bio-Plex 200 del sistema, las instrucciones del fabricante. En este protocolo están específicamente la medición de IL-1β, sin embargo, nosotros utilizamos este ensayo multiplex de citoquinas con el fin de medir simultáneamente múltiples citocinas en un ensayo con la ventaja de utilizar un pequeño volumen de muestra. </li></ol> 5. Resultados representante El análisis de la IL-1β de producción a la estimulación con LPS éxito y el ATP se muestra unasí la respuesta depende del número de células se estimularon. Hemos encontrado que x 2.0 10 6 células / ml es una concentración suficiente para producir una concentración óptima y medibles de IL-1β, así como otras citoquinas (Figura 2). Los ensayos de toda la sangre son útiles en el estudio de los pacientes que presentan manifestaciones autoinflamatoria. Estos pacientes pueden presentar un defecto en el sistema inmune innato, que se caracteriza por la sobreproducción de IL-1β (en comparación con los controles) a la estimulación con LPS solo (Figura 3). <img src="/files/ftp_upload/2662/2662fig1.jpg" alt="Figura 1" /> Figura 1. Imagen representativa de la tinción de AO de células nucleadas blancas de la sangre utilizada para determinar la concentración de células en muestras de sangre diluida. <img src="/files/ftp_upload/2662/2662fig2.jpg" alt="Figura 2" /> Figura 2. IL-1β producción medida en el sobrenadante de sangre de un donante sano a la estimulación con LPS en las concentraciones de células cada vez mayor. Las muestras fueron analizadas por triplicado. <img src="/files/ftp_upload/2662/2662fig3.jpg" alt="Figura 3" /> Figura 3. Los resultados representativos de la secreción de IL-1β medidos en los sobrenadantes de sangre total de dos controles normal y saludable, así como dos pacientes con IL-1β, de manifestaciones de autoinflamatoria.

<ol>
	<li>Martinon, F., Mayor, A., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasomes:+guardians+of+the+body.">The inflammasomes: guardians of the body.</a> Annu Rev Immunol. 27, 229-265 (2009).</li><li>Thurm, C. W., Halsey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytokine+production+using+whole+blood.">Measurement of cytokine production using whole blood.</a> Curr Protoc Immunol. 66, 7-18 .</li><li>Mariathasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopyrin+activates+the+inflammasome+in+response+to+toxins+and+ATP.">Cryopyrin activates the inflammasome in response to toxins and ATP.</a> Nature. 440, 228-232 (2006).</li><li>Gabay, C., Lamacchia, C., Palmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1+pathways+in+inflammation+and+human+diseases.">IL-1 pathways in inflammation and human diseases.</a> Nat Rev Rheumatol. 4, 232-241 (2010).</li><li>Franchi, L., Elgenbrod, T., Mu&#241;oz-Planillo, R., Nu&#241;ez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat Immunol. 3, 241-247 (2009).</li><li>Sims, J. E., Smith, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-1+family:+regulators+of+immunity.">The IL-1 family: regulators of immunity.</a> Nat Rev Immunol. 2, 89-102 (2010).</li><li>Haug, R., Jo&#248;, G. B., Westvik, A. B., Kierulf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+production+and+pattern+recognition+receptor+(PRR)+expression+in+whole+blood+stimulated+with+pathogen-associated+molecular+patterns+(PAMPs.">Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs.</a> Cytokine. 32, 304-315 (2005).</li><li>Netea, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+requirement+for+the+activation+of+the+inflammasome+for+processing+and+release+of+IL-1bin+monocytes+and+macrophages.">Differential requirement for the activation of the inflammasome for processing and release of IL-1bin monocytes and macrophages.</a> Blood. 113, 2324-2335 (2009).</li><li>Latz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasomes:+mechanisms+of+activation+and+function.">The inflammasomes: mechanisms of activation and function.</a> Curr Opin Immnol. 22, 28-33 (2010).</li><li>Masters, S. L., Simon, A., Aksentijevich, I., Kastner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horror+autoinflammaticus:+the+molecular+pathophysiology+of+autoinflammtory+disease.">Horror autoinflammaticus: the molecular pathophysiology of autoinflammtory disease.</a> Annu Rev Immunol. 27, 621-668 (2009).</li></ol>

La prueba de sangre entera se describe un método simple que reproduce mejor las condiciones fisiológicas en comparación con otras técnicas que requieren aislamiento de células y procesamiento de muestras extensas y la manipulación. Este método utiliza la visión Cellometer con AO para contar los glóbulos blancos en la sangre y por lo tanto, proporciona una manera de estandarizar la precisión muestras basadas en el número de células. Hay que tener en cuenta que para este ensayo, la...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Sodium Heparin Blood Collection Tubes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>366480</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 Media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>21870-100</td>
<td>&nbsp;</td>
<tr>
<td>Acridine Orange</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A3568</td>
<td>&nbsp;</td>
<tr>
<td>Cellometer Vision</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>&nbsp;</td>
<td>Contact company for instrument information</td>
</tr>
<tr>
<td>Cellometer Cell Counting Chambers</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>CHT4-SD100</td>
<td>&nbsp;</td>
<tr>
<td>24-well Tissue Culture Plate</td>
<td>&nbsp;</td>
<td>Corning Costar</td>
<td>3524</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Pure E. coli K12 LPS</td>
<td>&nbsp;</td>
<td>InvivoGen</td>
<td>tlrl-eklps</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#180;-tripohasphate disodium salt hydrate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A26209</td>
<td>&nbsp;</td>
<tr>
<td>Bio-plex 200 system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>Contact company for instrument information</td>
</tr>
<tr>
<td>Bio-Plex Pro human cytokine x-plex assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>x-plex assays are custom made according to specified cytokines of interest</td>
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accurate and simple measurement of the pro-inflammatory cytokine il-1&beta; using a whole blood stimulation assay

Los procesos inflamatorios como resultado de la secreción de mediadores solubles de las células inmunes, conducen a diversas manifestaciones en la piel, articulaciones y otros tejidos, así como la homeostasis alterada de citoquinas. El sistema inmune innato desempeña un papel crucial en el reconocimiento de patógenos y otros estímulos endógenos peligro. Una de las principales citoquinas liberadas por células del sistema inmune innato es interleuquina (IL) -1. Por lo tanto, utilizamos un análisis de sangre toda la estimulación con el fin de medir la secreción de citoquinas inflamatorias y, específicamente, de la citoquina pro-inflamatoria IL-1β 1, 2, 3. Los pacientes con trastornos genéticos del sistema inmune innato que causan síndromes de autoinflamatoria muestran una versión exagerada de IL-1β madura a la estimulación con LPS solo. Con el fin de evaluar el componente inmune innato de los pacientes que presentan patologías inflamatorias asociadas, se utiliza un inmunoensayo específico para detectar la respuesta inmune celular a patógenos patrones moleculares asociados (PAMP), tales como las endotoxinas bacterianas gram-negativas, el lipopolisacárido (LPS ). Estos PAMPs son reconocidos por receptores de reconocimiento de patógenos (RRP), que se encuentran en las células del sistema inmune innato 4, 5, 6, 7. Una señal de primaria, LPS, junto con una señal secundaria, ATP, es necesaria para la activación de la inflamasoma, un complejo multiproteico que los procesos de pro-IL-1β a su forma madura, bioactivos 4, 5, 6, 8, 9, 10. La prueba de sangre entera requiere una manipulación mínima de la muestra para evaluar la producción de citoquinas en comparación con otros métodos que requieren mucha mano de obra de aislamiento y cultivo de poblaciones celulares específicas. Este método se diferencia de otras pruebas de sangre entera estimulación, en lugar de diluir las muestras con una proporción de RPMI medios de comunicación, se realiza un recuento de glóbulos blancos directamente de la sangre entera diluida y, por tanto, estimular un número conocido de células blancas de la sangre en la cultura 2. Los resultados de este ensayo en sangre entera en particular demostrar una nueva técnica útil en el esclarecimiento de cohortes de pacientes que presentan fisiopatologías autoinflamatoria.

Watch this Scientific Journal Video about Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1Î² using a Whole Blood Stimulation Assay at JoVE.com

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

Se describe un inmunoensayo simple para medir la producción de citoquinas pro-inflamatorias, como IL-1 beta, en pacientes con fenotipos autoinflamatoria. Mediante la activación de las células en cultivos de sangre total con el patógeno patrones moleculares asociados, específicamente con lipopolisacárido, la secreción de citoquinas puede ser convenientemente evaluada en los sobrenadantes de sangre total.

La medición precisa y simple de la citoquina pro-inflamatoria IL-1β mediante un análisis de sangre entera estimulación

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

accurate-simple-measurement-pro-inflammatory-cytokine-il-1-using

Research

JoVE Journal

Medicine

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

26.2K vistas.  National Institute of Arthritis and Musculoskeletal and Skin Diseases. Se describe un inmunoensayo simple para medir la producción de citoquinas pro-inflamatorias, como IL-1 beta, en pacientes con fenotipos autoinflamatoria. Mediante la activación de las células en cultivos de sangre total con el patógeno patrones moleculares asociados, específicamente con lipopolisacárido, la secreción de citoquinas puede ser convenientemente evaluada en los sobrenadantes de sangre total.

Video: Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

La evaluación precisa y simple de las anastomosis vasculares en la placenta monocoriónicos con tinte de color

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Investigación

Medicina

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Medición de la presión intracraneal epidural en ratas, utilizando un transductor de presión de fibra óptica

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Rapid Point-of-Care Ensayo de la eficacia anticoagulante enoxaparina en sangre entera

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

Rápido y preciso exhalado Amoníaco Medición

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

En tiempo real Ensayos de citotoxicidad en sangre entera humana

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Un dispositivo sencillo de preparar rápidamente enteros Montes del Intestino Ratón

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry con estimulación cerebral profunda para estudiar la función cerebral normal y patológica

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

A largo plazo de medida de la presión arterial en ratones con libertad de movimientos mediante telemetría

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Evaluación de las propiedades anticoagulantes y antiinflamatorias de las células endoteliales mediante cultivo celular 3D y sangre no anticoagulada

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...