Cette recherche a été financée par le Programme de recherche intra-muros de l&#39;Institut national de l&#39;arthrite et les maladies musculo-squelettiques et la peau du National Institutes of Health.

1. Collecte de sang total <ol><li> Obtenir un minimum de 10 ml de sang périphérique de personnes touchées ainsi que l&#39;âge et le sexe témoins sains appariés. Le sang veineux doit être recueilli dans des tubes Vacutainer héparine sodique collecte de sang, plutôt que d&#39;utiliser d&#39;autres anticoagulants tels que l&#39;EDTA, et retourné plusieurs fois pour s&#39;assurer bon mélange avec l&#39;héparine de sodium. Notez que 10 ml est le volume minimum de sang total requis pour les échantillons en double plaquage pour ce dosage particulier. </li><li> Les échantillons doivent être traités dès que possible après le prélèvement pour obtenir les meilleurs résultats. Toutefois, le sang hépariné peuvent être stockés à température ambiante sous un faible éclairage, jusqu&#39;à 24 heures jusqu&#39;à ce que prêt à être traité (2). </li></ol> 2. Préparation de sang total <ol><li> Centrifuger tous les échantillons de sang à 3000 rpm (avec frein désactivé) pendant dix minutes à température ambiante. Sans déranger la couche leucocytaire, le plasma peut être recueilli par le haut et stockées à d&#39;autres fins expérimentales. Les échantillons de plasma peuvent être stockés temporairement à -20 ° C ou -80 ° C pour le stockage à long terme </li><li> Une fois que le plasma est recueilli, laver les échantillons par remise en suspension des globules rouges (RBC) et la couche leuco granulés avec du sérum libre (incomplète) du milieu RPMI 1640 (à température ambiante). Délicatement la pipette le mélange dans les tubes de prélèvement et le transfert d&#39;un tube de 50 ml conique. </li><li> Volume inférieur à 50 mL avec les médias RPMI incomplète de la culture 1640 et retourner doucement les tubes afin de créer un mélange homogène. Centrifuger les échantillons à 3000 rpm (avec frein désactivé) pendant dix minutes. Faire en sorte il ya une séparation claire, aspirer le surnageant, en faisant attention à ne pas perturber la couche leucocytaire. Répétez cette procédure 2-4 fois pour s&#39;assurer que tous les contaminants et les cytokines liés sont retirés avant la stimulation. </li><li> Après le dernier lavage, resuspendre le culot de RBC et de buffy coat sorte que le volume total est de 20 mL (pour 10 ml de sang total initialement établi) avec incomplète du milieu RPMI 1640. Le volume total peut être ajusté en fonction du volume de sang initialement élaboré. </li><li> Utilisation de la Vision Cellometer, sang dilué peut être compté, sans globules rouges lyse en choisissant l&#39;option de globules blancs comptent utilisant l&#39;acridine orange (AO). Ajouter un seul sang, une partie dilué avec un AO partie et charge de 20 uL du mélange 1:1 dans une chambre de comptage à usage unique. La concentration finale de AO après dilution devrait être 1μg/mL. </li><li> Ajuster en conséquence avec incomplète du milieu RPMI 1640 à une concentration cellulaire finale de 2,0 x 10 6 cellules / ml. </li></ol> 3. La stimulation du sang total <ol><li> Le dosage nécessite un minimum de quatre conditions (chacun fait en double exemplaire): non stimulées, l&#39;ajout de LPS seul, l&#39;ajout d&#39;ATP seul, et enfin, plus LPS ATP additionnées. Ajouter 1 mL de l&#39;2,0 préparés x 10 6 cellules / ml de sang diluée dans l&#39;ensemble bien appropriés d&#39;une plaque de 24 puits de culture de tissu. </li><li> Les stimulants doit être reconstitué et mis à la température ambiante avant de commencer l&#39;essai de stimulation. Lyophilisée LPS et l&#39;ATP doit être reconstitué selon les instructions du fabricant. Après reconstitution, le LPS et l&#39;ATP peut être encore diluée à une concentration correcte des stocks en utilisant incomplète du milieu RPMI 1640. </li><li> Ajouter au LPS LPS seule aussi bien que le LPS, plus l&#39;ATP ainsi afin que la concentration de travail finale du LPS est 1 ug / ml. Le temps de stimulation totale est de 3 heures. Cependant, le sang entier est stimulé par le LPS pendant 2 heures et 40 minutes. Les échantillons doivent être placés dans une étuve à 37 ° C et 5% de CO 2. </li><li> L&#39;ATP est ajoutée au cours des 20 dernières minutes de la période de trois heures de stimulation. Ajouter de l&#39;ATP aux seuls ATP et le LPS et le bien de l&#39;ATP et des échantillons dans l&#39;incubateur. La concentration finale de travail de l&#39;ATP est de 2mm. </li><li> Après la période d&#39;incubation de 3 heures, recueillir surnageants par aliquotage chaque échantillon dans un tube de 1,5 ml et ensuite centrifugation des échantillons à 10000 rpm pendant 2 minutes à température ambiante. Transférer les surnageants à un autre ensemble de 1,5 microtubes. </li><li> Les échantillons peuvent être analysés immédiatement ou stockées temporairement à -20 ° C ou -80 ° C pour stockage à long terme. </li></ol> 4. Assay cytokines <ol><li> Les échantillons qui sont congelés doivent s&#39;équilibrer à température ambiante avant l&#39;analyse. Les concentrations de cytokines IL-1βcan être évalués en utilisant une variété de méthodes. Nous utilisons la Bio-Plex cytokine pro-Plex humaine x dosage avec le Bio-Plex 200 système, selon les instructions du fabricant. Dans ce protocole, nous sommes particulièrement l&#39;IL-1β mesure, cependant, nous utilisons ce test multiplex de cytokines dans le but de mesurer simultanément multiples cytokines dans un essai avec l&#39;avantage d&#39;utiliser un petit volume d&#39;échantillon. </li></ol> 5. Les résultats représentatifs Analyse de l&#39;IL-1β de production lors de la stimulation de succès avec le LPS et l&#39;ATP ne montre unesoi la réponse dépend du nombre de cellules étant stimulée. Nous avons constaté que 2,0 x 10 6 cellules / ml est une concentration suffisante pour produire une concentration optimale et mesurable de l&#39;IL-1β ainsi que d&#39;autres cytokines (figure 2). Dosages de sang total sont utiles dans l&#39;étude les patients qui présentent des manifestations auto-inflammatoires. Ces patients peuvent présenter un défaut dans le système immunitaire inné, ce qui peut être caractérisée par la surproduction d&#39;IL-1β (comparativement aux témoins) après stimulation par le LPS seul (figure 3). <img src="/files/ftp_upload/2662/2662fig1.jpg" alt="Figure 1" /> Figure 1. Image représentative de la coloration AO pour nucléées globules blancs utilisés afin de déterminer la concentration de cellules dans le nombre dilué échantillons de sang total. <img src="/files/ftp_upload/2662/2662fig2.jpg" alt="Figure 2" /> Figure 2. IL-1β production mesurée dans les surnageants de sang total d&#39;un donneur sain après stimulation par le LPS à des concentrations de cellules augmente. Les échantillons ont été dosés en triple exemplaire. <img src="/files/ftp_upload/2662/2662fig3.jpg" alt="Figure 3" /> Figure 3. Les résultats représentatifs de l&#39;IL-1β sécrétion mesurée dans les surnageants de sang entier à partir de deux témoins sains normaux, ainsi que deux patients se présentant avec de l&#39;IL-1β associée manifestations auto-inflammatoires.

<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>

Le dosage sanguin de l&#39;ensemble décrit est une méthode simple qui se rapproche le plus des conditions physiologiques par rapport aux autres techniques qui nécessitent l&#39;isolement cellulaire et de traitement des échantillons vaste et la manipulation. Cette méthode utilise la Vision Cellometer avec AO pour compter les globules blancs dans le sang et fournit donc un moyen de normaliser les échantillons avec précision en fonction du nombre de cellules. Il est à noter que pour ce ...

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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>
		</tr>
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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

Les processus inflammatoires résultant de la sécrétion de médiateurs solubles par les cellules immunitaires, conduire à diverses manifestations dans la peau, les articulations et autres tissus ainsi que l&#39;homéostasie des cytokines modifiées. Le système immunitaire inné joue un rôle crucial dans la reconnaissance des pathogènes et d&#39;autres stimuli endogènes de danger. Une des principales cytokines libérées par les cellules immunitaires innées est l&#39;interleukine (IL) -1. Par conséquent, nous utilisons un dosage sanguin de l&#39;ensemble de stimulation afin de mesurer la sécrétion de cytokines inflammatoires et plus particulièrement de l&#39;cytokine pro-inflammatoire IL-1β 1, 2, 3. Les patients atteints de dysfonctionnements génétiques du système immunitaire inné causant syndromes auto-inflammatoires montrent une libération exagérée de l&#39;IL-1β matures lors de la stimulation avec le LPS seul. Afin d&#39;évaluer la composante immunitaire inné des patients qui présentent des pathologies associées inflammatoires, nous utilisons un immunodosage spécifique pour détecter les réponses immunitaires cellulaires aux agents pathogènes motifs moléculaires associés (PAMP), tels que l&#39;endotoxine bactérienne gram-négatif, le lipopolysaccharide (LPS ). Ces PAMPs sont reconnus par des récepteurs de reconnaissance des pathogènes (PRR), qui se trouvent sur ​​les cellules du système immunitaire inné 4, 5, 6, 7. Un signal fort, LPS, en conjonction avec un signal secondaire, l&#39;ATP, est nécessaire pour l&#39;activation de l&#39;inflammasome, un complexe multiprotéique que les processus pro-IL-1β à sa maturité, forme bioactive 4, 5, 6, 8, 9, 10. Le dosage sanguin de l&#39;ensemble nécessite une manipulation minimale de l&#39;échantillon pour évaluer la production de cytokines par rapport à d&#39;autres méthodes qui nécessitent une isolation de main-d&#39;oeuvre et de la culture des populations de cellules spécifiques. Cette méthode diffère des autres toute la stimulation des dosages sanguins; plutôt que de diluer les échantillons avec un ratio de RPMI médias, nous réalisons un nombre de globules blancs du sang total directement dilué et donc, de stimuler un nombre connu de globules blancs dans la culture 2. Les résultats de ce dosage sanguin toute particulière de démontrer une nouvelle technique utile dans l&#39;élucidation des cohortes de patients présentant des physiopathologies auto-inflammatoires.

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

Nous décrivons une méthode de dosage immunologique pour mesurer la production de cytokines pro-inflammatoires, tels que la production d&#39;IL-1 bêta, chez les patients présentant des phénotypes auto-inflammatoires. En activant les cellules dans les cultures de sang total avec des agents pathogènes motifs moléculaires associés, en particulier avec les lipopolysaccharides, la sécrétion de cytokines peuvent être facilement évaluée dans les surnageants de sang total.

Mesure précise et simple de la cytokine pro-inflammatoire IL-1β en utilisant un dosage de sang total Stimulation

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 vues.  National Institute of Arthritis and Musculoskeletal and Skin Diseases. Nous décrivons une méthode de dosage immunologique pour mesurer la production de cytokines pro-inflammatoires, tels que la production d'IL-1 bêta, chez les patients présentant des phénotypes auto-inflammatoires. En activant les cellules dans les cultures de sang total avec des agents pathogènes motifs moléculaires associés, en particulier avec les lipopolysaccharides, la sécrétion de cytokines peuvent être facilement évaluée dans les surnageants de sang t...

Vidéo: 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.

Évaluation précise et simple des anastomoses vasculaires dans le placenta monochoriales utilisant colorant

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 ...

Recherche

Médecine

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.

Péridurale Mesure de la pression intracrânienne chez le rat en utilisant un transducteur de pression à fibre optique

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 Test de l&#39;efficacité anticoagulante énoxaparine dans le sang total

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.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

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.

Rapide et précis expiré Breath ammoniac mesure

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 temps réel cytotoxicité dans le sang total humain

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 dispositif simple d&#39;élaborer rapidement entiers Monts de l&#39;intestin de souris

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.

Utilisation de Saccadometry avec la stimulation cérébrale profonde pour l&#39;étude normale et pathologique fonction cérébrale

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.

Mesure la pression artérielle à long terme chez la souris Librement mobile à l&#39;aide de télémesure

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.

Évaluation des propriétés anticoagulantes et anti-inflammatoires de cellules endothéliales à l’aide de la Culture cellulaire 3D et sang total Non-anticoagulant

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