Esta pesquisa foi apoiada pelo Programa de Pesquisa Intramural do Instituto Nacional de Artrite e Doenças músculo-esqueléticas e da pele do National Institutes of Health.

1. Recolha de Sangue Total <ol><li> Obter um mínimo de 10 mL de sangue periférico de indivíduos afectados, bem como idade e sexo pareados controles saudáveis. Sangue venoso deve ser coletado em heparina sódica tubos vacutainer de coleta de sangue, ao invés de usar outros anticoagulantes, como EDTA, e invertido várias vezes para garantir a mistura adequada com a heparina sódica. Note-se que 10 mL é o volume mínimo de sangue necessário para amostras de revestimento em duplicado para este ensaio particular. </li><li> As amostras devem ser processadas o mais rapidamente possível após a coleta para obter melhores resultados. No entanto, o sangue heparinizado pode ser armazenado em temperatura ambiente sob iluminação fraca por até 24 horas até que esteja pronto para ser processado (2). </li></ol> 2. Preparação de sangue total <ol><li> Centrífuga todas as amostras de sangue a 3.000 rpm (com freio desligado) por dez minutos em temperatura ambiente. Sem perturbar o buffy coat, o plasma pode ser recolhida a partir do topo e armazenados para outros fins experimentais. As amostras de plasma podem ser temporariamente armazenados a -20 ° C ou -80 ° C para o armazenamento de longo prazo </li><li> Uma vez que plasma é coletado, lave amostras de ressuspender a célula vermelha do sangue (RBC) e pellet buffy coat com soro livre (incompleta) RPMI 1640 media (à temperatura ambiente). Pipeta suavemente a mistura nos tubos de coleta e transferência para um tubo cônico de 50 mL. </li><li> Volume até 50 mL com incompleta RPMI 1640 meios de cultura e inverter cuidadosamente os tubos para criar uma mistura homogénea. Centrifugar as amostras a 3000 rpm (com freio desligado) por dez minutos. Certificando-se que há uma separação clara, aspirar o sobrenadante, tomando cuidado para não perturbar a buffy coat. Repita este procedimento 2-4 vezes para garantir que todos os contaminantes e citocinas vinculados são removidos antes da estimulação. </li><li> Após a última lavagem, ressuspender o sedimento RBC e buffy coat de modo que o volume total é de 20 mL (para 10 mL de sangue total inicialmente sorteado) com incompleta RPMI 1640 mídia. O volume total pode ser ajustado com base no volume de sangue inicialmente sorteado. </li><li> Usando a Visão Cellometer, o sangue diluído pode ser contado sem hemácias lise escolhendo a opção de leucócitos contagem de células usando laranja de acridina (AO). Adicione uma parte de sangue diluída com um AO parte e carga de 20 mL da mistura 1:1 em uma câmara de contagem descartáveis. A concentração final de AO após a diluição deve ser 1μg/mL. </li><li> Ajustar de acordo com incompleta RPMI 1640 mídia para uma concentração celular final de 2,0 x 10 6 células / mL. </li></ol> 3. Estimulação do sangue total <ol><li> O ensaio requer um mínimo de quatro condições (cada feito em duplicado): não estimuladas, a adição de LPS sozinho, a adição de ATP por si só, e, finalmente, LPS, mais ATP somados. Adicionar 1 mL do preparado 2,0 x 10 6 células / mL de sangue diluído inteira dentro do poço apropriado de uma placa de cultura de 24 poços de tecido. </li><li> Os estimulantes deve ser reconstituído e trouxe até à temperatura ambiente antes de iniciar o ensaio de estimulação. Liofilizado LPS e ATP deve ser reconstituído de acordo com as instruções do fabricante. Após a reconstituição, LPS e ATP pode ser diluída a uma concentração de estoque adequado usando incompleta RPMI 1640 mídia. </li><li> Adicionar ao LPS LPS só, bem como para a LPS, mais ATP bem para que a concentração final de trabalho de LPS é de 1 mg / mL. O tempo de estimulação total é de 3 horas. No entanto, o sangue total é estimulado com LPS por 2 horas e 40 minutos. As amostras devem ser colocados em uma incubadora a 37 ° C e 5% CO 2. </li><li> ATP é adicionado durante os restantes 20 minutos do período de estimulação de 3 horas. Adicionar ao ATP ATP por si só e os LPS e ATP bem e amostras lugar na incubadora. A concentração final de trabalho de ATP é 2mM. </li><li> Após o período de incubação de 3 horas, recolher sobrenadantes de alíquotas de cada amostra num tubo de microcentrífuga de 1,5 mL e, posteriormente, centrifugar amostras de 10000 rpm por 2 minutos em temperatura ambiente. Transferir o sobrenadante para outro conjunto de tubos de 1,5 microcentrífuga. </li><li> As amostras podem ser analisadas imediatamente ou armazenados temporariamente a -20 ° C ou -80 ° C para armazenamento de longo prazo. </li></ol> 4. Ensaio de citocinas <ol><li> Amostras que são congelados necessidade de atingir a temperatura ambiente antes da análise. Concentrações das citocinas IL-1βcan para ser avaliado usando uma variedade de métodos. Nós utilizamos o Bio-plex citocina Pro humana x-plex ensaio com o Bio-Plex 200 sistema, por instruções do fabricante. Neste protocolo que são especificamente medição IL-1β, no entanto, nós utilizamos neste ensaio multiplex de citocinas, a fim de medir simultaneamente múltiplas citocinas em um ensaio com a vantagem de usar um volume de amostra pequena. </li></ol> 5. Resultados representante Análise de IL-1β produção com a estimulação de sucesso com LPS e ATP mostra um fazersi a resposta depende do número de células sendo estimulada. Descobrimos que 2,0 x 10 6 células / mL é uma concentração suficiente para produzir uma concentração ótima e mensurável de IL-1β, assim como outras citocinas (Figura 2). Doseamentos de sangue total são úteis no estudo de pacientes que se apresentam com manifestações Autoinflamatórias. Esses pacientes podem apresentar um defeito no sistema imune inato, que pode ser caracterizada pela superprodução de IL-1β (quando comparados aos controles) após estimulação com LPS isoladamente (Figura 3). <img src="/files/ftp_upload/2662/2662fig1.jpg" alt="Figura 1" /> Figura 1. Image Representante do AO coloração para nucleadas células brancas do sangue utilizado para determinar a concentração de células em amostras de sangue total diluído. <img src="/files/ftp_upload/2662/2662fig2.jpg" alt="Figura 2" /> Figura 2. Produção de IL-1β medido em sobrenadantes de sangue total de um doador saudável com a estimulação com LPS em concentrações crescentes de células. As amostras foram analisadas em triplicata. <img src="/files/ftp_upload/2662/2662fig3.jpg" alt="Figura 3" /> Figura 3. Resultados Representante da IL-1β secreção medido em sobrenadantes de sangue total a partir de dois saudáveis ​​controles normais, assim como duas pacientes com IL-1β associada manifestações Autoinflamatórias.

<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>

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O ensaio de sangue total descrito é um método simples que reproduz com mais exatidão condições fisiológicas quando comparada a outras técnicas que exigem isolamento de células e processamento das amostras extensas e manipulação. Este método utiliza a Visão Cellometer com AO a contagem de células brancas do sangue em sangue total e, portanto, fornece uma maneira de padronizar as amostras com precisão com base em números de celular. É de notar que, para este ensaio, o sangue to...

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<td>Sodium Heparin Blood Collection Tubes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>366480</td>
<td>&nbsp;</td>
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<td>RPMI 1640 Media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
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<td>&nbsp;</td>
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<td>Acridine Orange</td>
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<td>Invitrogen</td>
<td>A3568</td>
<td>&nbsp;</td>
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<td>Cellometer Vision</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>&nbsp;</td>
<td>Contact company for instrument information</td>
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<tr>
<td>Cellometer Cell Counting Chambers</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>CHT4-SD100</td>
<td>&nbsp;</td>
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<td>24-well Tissue Culture Plate</td>
<td>&nbsp;</td>
<td>Corning Costar</td>
<td>3524</td>
<td>&nbsp;</td>
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<td>Ultra Pure E. coli K12 LPS</td>
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<td>Adenosine 5&#180;-tripohasphate disodium salt hydrate</td>
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<td>Bio-plex 200 system</td>
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<td>Bio-Plex Pro human cytokine x-plex assay</td>
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<td>Bio-Rad</td>
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accurate and simple measurement of the pro-inflammatory cytokine il-1&beta; using a whole blood stimulation assay

Processos inflamatórios resultantes da secreção de mediadores solúveis por células do sistema imunológico, levar a várias manifestações na pele, articulações e outros tecidos, bem como a homeostase das citocinas alterada. O sistema imune inato desempenha um papel crucial no reconhecimento de patógenos e outros estímulos endógenos perigo. Uma das principais citocinas liberado por células do sistema imunológico inato é Interleucina (IL) -1. Portanto, nós utilizamos um ensaio de estimulação todo sangue para medir a secreção de citocinas inflamatórias e especificamente da citocinas pró-inflamatórias IL-1β 1, 2, 3. Pacientes com disfunções genéticas do sistema imune inato causando síndromes Autoinflamatórias mostram uma liberação exagerada de IL 1β madura-upon estimulação com LPS sozinho. Para avaliar o componente imunológico inato de pacientes que apresentam patologias inflamatórias associadas, usamos um imunoensaio específico para detectar as respostas imunes celulares ao patógeno-padrões moleculares associados (PAMPs), tais como a endotoxina bacteriana gram-negativa, o lipopolissacarídeo (LPS ). Estes PAMPs são reconhecidos por receptores de reconhecimento de patógenos (PRRs), que são encontrados nas células do sistema imune inato 4, 5, 6, 7. Um sinal primário, LPS, em conjunto com um sinal secundário, ATP, é necessária para a ativação do inflammasome, um complexo multiprotein que os processos pró-IL-1β à sua forma, maduro bioativos 4, 5, 6, 8, 9, 10. O ensaio de sangue total requer a manipulação de amostra mínima para avaliar a produção de citocinas, quando comparado a outros métodos que exigem o isolamento trabalho intensivo e cultura de populações de células específicas. Este método difere de outros ensaios de estimulação de sangue total, ao invés de diluir as amostras com uma proporção de RPMI mídia, realizamos uma contagem de células brancas do sangue diretamente do sangue total diluído e, portanto, estimular um número conhecido de células brancas do sangue na cultura 2. Os resultados deste ensaio de sangue especial toda demonstrar uma nova técnica útil na elucidação coortes paciente apresentando fisiopatologias Autoinflamatórias.

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

Nós descrevemos um imunoensaio simples para medir a produção de citocinas pró-inflamatórias, como IL-1 beta de produção, em pacientes com fenótipos Autoinflamatórias. Pela ativação de células em culturas de sangue total com patógeno-padrões moleculares associados, especificamente com lipopolissacarídeo, a secreção de citocinas podem ser convenientemente avaliados em sobrenadantes de sangue total.

Medição precisa e simples do pró-inflamatórias IL-1β citocinas usando um ensaio Estimulação Sangue Total

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

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Research

JoVE Journal

Medicine

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

26.2K visualizações.  National Institute of Arthritis and Musculoskeletal and Skin Diseases. Nós descrevemos um imunoensaio simples para medir a produção de citocinas pró-inflamatórias, como IL-1 beta de produção, em pacientes com fenótipos Autoinflamatórias. Pela ativação de células em culturas de sangue total com patógeno-padrões moleculares associados, especificamente com lipopolissacarídeo, a secreção de citocinas podem ser convenientemente avaliados em sobrenadantes de sangue total.

Vídeo: 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.

Avaliação precisas e simples de anastomoses vasculares em Placenta monocoriônicas usando Dye Colorido

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 ...

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.

Medida da Pressão Intracraniana peridural em ratos, utilizando um transdutor de pressão 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.

Ensaio de Point-of-Care rápida de eficácia anticoagulante enoxaparina em sangue 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.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de 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.

Medição Amônia ar exalado Rápido e Preciso

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.

Em tempo real de Citotoxicidade em Sangue Total Humano

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.

Um dispositivo simples de preparar rapidamente inteiras Mounts do mouse Intestino

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 com Estimulação Cerebral Profunda para estudar normal e patológico função cerebral

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.

Longo prazo medição de pressão arterial em ratos movimentando-se livremente utilizar telemetria

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.

Avaliação das propriedades das células endoteliais utilizando cultura de células 3D e sangue não anticoagulado anticoagulantes e anti-inflamatório

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