Agradecemos a Virginia Doherty y Junmei Yao para la asistencia técnica. Esta investigación fue financiada por el NIMH (R01 MH084966), y la Oficina de Investigación del Ejército de Estados Unidos y la Agencia de Proyectos de Investigación Avanzada de Defensa (conceder W911NF-10-1-0059) a KAG.

Todos los experimentos se realizaron utilizando adulto ratas macho Long-Evans. Todos los procedimientos fueron de acuerdo con los Institutos Nacionales de Salud (NIH) Guía para el Cuidado y Uso de Animales de Laboratorio y fueron aprobados por el Comité Institucional Cuidado de Animales y el empleo del Instituto de Tecnología de Massachusetts y el Cuidado de Animales y el Empleo Oficina de Revisión de el USAMRMC. 1. Preparación <ol><li> Heparinise el catéter y la jeringa mediante la colocación de la aguja blindado en un tubo que contenía heparina 500 l (1.000 unidades USP / ml) y, a continuación aspirando y expulsando solución de heparina a través de la aguja. <ol><li> Adjuntar un catéter de mariposa a la jeringa. Mantenga el escudo sobre la aguja del catéter para proteger la punta afilada de los daños. </li><li> Retirar un volumen de heparina que es ligeramente mayor que el volumen de sangre que se recoge. Separe la jeringa y llenarlo con aire. </li><li> Vuelva a colocar la jeringa al catéter y utilizar elde aire para expulsar el exceso de solución de heparina; garantizan solamente trazas permanecen en los tubos, agujas y jeringas. </li><li> Coloque el catéter estéril, con la jeringa todavía unida, sobre una superficie estéril. </li></ol></li><li> Asegurar rápidamente la rata en un paño limpio asegurando que delanteras y patas traseras se encuentran en una posición cómoda y la respiración es irrestricta. <ol><li> Asegure la envoltura con gancho y lazo de cinta; asegurar que los genitales externos no están constreñidos. </li><li> Tener un ayudante con suavidad y firmeza frenar la rata (abdomen y base de la cola) sobre una superficie sólida con la cola colgando del borde del mostrador. </li></ol></li></ol> Muestreo 2. Sangre <ol><li> Sumerja la cola de 42 ° C el agua para 40 a 50 segundos para dilatar los vasos sanguíneos y seque la cola con una toalla de papel. Localice la vena de la cola para ser desangrado (girar todo el cuerpo con la cola para evitar torcer la cola). Nota: el calentamiento suficiente de la cola es crítico para la rápida collection de una muestra de sangre. Si se estrecha la vasculatura, la colocación apropiada del catéter es difícil, y el flujo sanguíneo se reduce enormemente. Una almohadilla de calentamiento puede ser utilizado como una alternativa a la inmersión en agua. </li><li> Identificar el punto de muestreo. Nota: La arteria se encuentra a lo largo del aspecto mediados de dorsal de la cola; no utilice este para el muestreo. <ol><li> Objetivo, ya sea las venas de la cola izquierda y derecha que se encuentran lateral a la arteria. La pigmentación de la cola, que varía por la tensión y aumenta con la edad, puede oscurecer algunos de la vasculatura. Objetivo a una porción de la vena en la parte inferior de la cola. </li><li> Limpie el área objetivo con clorhexidina al 2% solución antiséptica. </li></ol></li><li> Crear una presión negativa en la jeringa y el catéter al retirar el émbolo de cero a aproximadamente 50 l. </li><li> Mantenga la cola suave y firmemente cerca de la punta para mantener la cola recta durante la recogida de muestras. Asegúrese de que el flujo sanguíneo no está ocluida por un agarre demasiado apretado.</li><li> Lentamente insertar el catéter en la vena en un ángulo superficial de aproximadamente 5 cm de la punta de la cola. Cuando se penetra la vena, la sangre fluirá en el catéter. Retire lentamente el émbolo de la jeringa para recoger el volumen deseado a un ritmo constante (~ 20 l por segundo). <ol><li> Consulte al personal veterinario para obtener información sobre el volumen de sangre máxima que se puede recoger. La cantidad máxima de sangre que debe ser recogido depende del estado de peso y la salud de la rata. No retirar más de 15% del volumen total de sangre en un período de 14 días. Nota: La sangre es mucho más difícil de cobrar a los animales que se destacaron de forma aguda en los minutos previos a la recogida de muestras, porque las hormonas del estrés constriñen los vasos. Por ejemplo, mover jaula de la rata a una habitación novela, teniendo varios minutos para envolver el animal, o inserción repetida del catéter en una vena son todos propensos a desencadenar una respuesta de estrés agudo. </li><li> Facilitar blooflujo d por &quot;ordeñar&quot; la vena. Ejecutar un dedo a lo largo de la longitud de la vena, desde la base hacia la punta de la cola, pero siguen siendo más de 2 cm desde la punta de la aguja que se inserta el catéter o puede desprenderse de la vena. </li><li> Si la sangre no puede ser recogido con éxito desde el sitio inicial de la penetración del catéter, vuelva a insertar la aguja más arriba en la vena. Si la sangre se recogió en el lugar inicial, re-presurizar la aguja desconectando y volviendo a conectar el catéter y la jeringa antes de la re-inserción en la vena. En general, evitar las penetraciones adicionales. </li><li> Como múltiples penetraciones pueden causar colapso vena de la cola, en el que el suministro de sangre a la cola se corta y el tejido blando de la cola se vuelve necrotizado, la eutanasia la rata si hay cola colapso vena. </li></ol></li><li> Cuando se recoge el volumen de muestra adecuado, liberar la presión en la jeringa al desconectar y volver a conectar el catéter. Aspirar ligeramente mediante el émbolo de la jeringa (~ 50 &amp; #181; l), y retirar la aguja de la vena. Nota: Si se retira la aguja sin primero liberando la presión en la jeringa, la sangre gotea desde la aguja. </li><li> Brevemente aplique presión en el sitio de inserción para detener el sangrado, y limpie el área con solución antiséptica. Regreso a la rata a su jaula. </li></ol> 3. Procesamiento de la muestra de sangre <ol><li> Aspirar el aire para asegurar que no sangre permanece dentro de la aguja del catéter, y el uso de las tijeras para cortar el tubo del catéter justo por encima de la aguja. Expulsar la sangre en un tubo de microcentrífuga de 1,5 ml estéril. Nota: Si la sangre es empujada a través de la aguja, la fuerza de cizallamiento puede causar que las células rojas de la sangre a la ruptura que puede interferir con muchos ensayos aguas abajo. Retire la aguja para evitar la hemólisis. <ol><li> Para recoger el plasma sanguíneo, las trompas de uso que contienen EDTA como anticoagulante (en este caso, utilice 10 l de EDTA 0,1 M de 200 a 400 l de sangre; garantizar la concentración de EDTA empleada no interfiere won el ensayo de aguas abajo) y el lugar en el hielo. <ol><li> Girar muestras de sangre entera a 2100 xg en una centrífuga refrigerada (4 ° C) durante 10 min a menos de 10 min de la recolección. Eluir el plasma, evitando molestar a las capas de glóbulos rojos y blancos. </li></ol></li><li> Para recoger el suero sanguíneo, lugar muestras (sin anticoagulante) a temperatura ambiente durante un máximo de 30 minutos para permitir la coagulación. Haga girar los tubos de recogida en una centrífuga refrigerada (4 ° C) a 2000 x g. El suero puede entonces ser eluido. </li></ol></li><li> Use muestras de inmediato, o tienda a -80 ° C durante un máximo de un año. </li></ol>

<ol>
	<li>Vausort, M., Wagner, D. R., Devaux, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Noncoding+RNAs+in+Patients+with+Acute+Myocardial+Infarction.">Long Noncoding RNAs in Patients with Acute Myocardial Infarction.</a> Circ Res. 115 (7), 668-677 (2014).</li><li>Shah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+for+Early+Detection+of+Colorectal+Cancer+and+Polyps:+Systematic+Review.">Biomarkers for Early Detection of Colorectal Cancer and Polyps: Systematic Review.</a> Cancer Epidemiol Biomarkers Prev. 23 (9), 1712-1728 (2014).</li><li>Chan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+blood-based+protein+biomarker+strategies+in+the+study+of+psychiatric+disorders.">Applications of blood-based protein biomarker strategies in the study of psychiatric disorders.</a> Prog Neurobiol. , (2014).</li><li>Cao, L., Li, L., Lin, D., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+learning+impairment+that+is+mediated+by+interleukin+1beta+in+rodents.">Isoflurane induces learning impairment that is mediated by interleukin 1beta in rodents.</a> PLoS One. 7 (12), e51431 (2012).</li><li>Culley, D. J., Baxter, M. G., Yukhananov, R., Crosby, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impairment+of+acquisition+of+a+spatial+memory+task+following+isoflurane-nitrous+oxide+anesthesia+in+rats.">Long-term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats.</a> Anesthesiology. 100 (2), 309-314 (2004).</li><li>Vahl, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+ACTH+and+corticosterone+sampling+methods+in+rats.">Comparative analysis of ACTH and corticosterone sampling methods in rats.</a> Am J Physiol Endocrinol Metab. 289 (5), E823-E828 (2005).</li><li>Kalinichenko, L. S., Koplik, E. V., Pertsov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+peripheral+blood+in+rats+with+various+behavioral+characteristics+during+acute+emotional+stress.">Cytokine profile of peripheral blood in rats with various behavioral characteristics during acute emotional stress.</a> Bull Exp Biol Med. 156 (4), 441-444 (2014).</li><li>McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+stress+hormones+in+health+and+disease:+Understanding+the+protective+and+damaging+effects+of+stress+and+stress+mediators.">Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators.</a> Eur J Pharmacol. 583 (2-3), 174-185 (2008).</li><li>Sanchez, A., Toledo-Pinto, E. A., Menezes, M. L., Pereira, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+norepinephrine+and+epinephrine+concentrations+in+adrenal+gland+of+the+rats+submitted+to+acute+immobilization+stress.">Changes in norepinephrine and epinephrine concentrations in adrenal gland of the rats submitted to acute immobilization stress.</a> Pharmacol Res. 48 (6), 607-613 (2003).</li><li>Meyer, R. M., Burgos-Robles, A., Liu, E., Correia, S. S., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ghrelin-growth+hormone+axis+drives+stress-induced+vulnerability+to+enhanced+fear.">A ghrelin-growth hormone axis drives stress-induced vulnerability to enhanced fear.</a> Mol Psychiatry. , (2013).</li><li>Knoll, A. T., Carlezon, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynorphin,+stress,+and+depression.">Dynorphin, stress, and depression.</a> Brain Res. 1314 (56-73), (2010).</li><li>Harvey, B. H., Brand, L., Jeeva, Z., Stein, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical/hippocampal+monoamines,+HPA-axis+changes+and+aversive+behavior+following+stress+and+restress+in+an+animal+model+of+post-traumatic+stress+disorder.">Cortical/hippocampal monoamines, HPA-axis changes and aversive behavior following stress and restress in an animal model of post-traumatic stress disorder.</a> Physiol Behav. 87 (5), 881-890 (2006).</li></ol>

Los autores no tienen nada que revelar.

Aquí se describe un procedimiento rápido y sencillo para la obtención de una muestra de sangre de una rata que ofrece importantes ventajas sobre otras técnicas de uso común. En primer lugar, no requiere anestesia, en contraste con el muestreo de la vena yugular o seno retroorbital. Cuando se recogen muestras de sangre que rodea procedimientos conductuales, la administración de anestésicos no es deseable porque puede interferir con el aprendizaje y 4,5 memoria. En segundo lugar, ofrece la posibilidad de recoger volúmenes de sangre más grandes que otras técnicas de venopunción, como la recogida de las venas safena pedal o dorsales. Usando la técnica descrita aquí, hasta 1,5 ml de sangre se puede recoger de una rata en un solo punto de tiempo, un volumen que permite fácilmente múltiples ensayos que se ejecutan en paralelo. Por último, este procedimiento minimiza el potencial de daño a los tejidos en comparación con la amputación punta de la cola o sangrado retroorbital. El uso de este procedimiento facilita el cumplimiento del Animal WLey elfare y la Guía para el Cuidado y Uso de Animales de Laboratorio, el cual requiere reducir al mínimo el dolor y la angustia que resultan de procedimientos de laboratorio realizadas en los animales. Se recomienda que los investigadores nuevos para este método practican las técnicas de inmovilización y de sangrado de la cola con el fin de minimizar el tiempo que los animales experimentales son restringidos. La sangre recogida en menos de 3 minutos desde el inicio de la restricción proporciona resultados óptimos. El protocolo descrito aquí puede utilizarse para el muestreo de 1 a 4 veces por semana, pero no más de dos veces por día. Mientras que las colecciones de sangre repetidas pueden llevar a cabo, diferentes sitios de muestreo que se mueven hacia arriba desde la base de la cola deben ser utilizados, y las venas de la cola izquierda y derecha deben alternarse como sitios de muestreo. El volumen total de sangre de los roedores es 6-7% de su peso corporal, y no más de 15% del volumen total de la sangre se debe recoger dentro de un período de 2 semanas. Sueroo plasma comprende aproximadamente un 40-60% del volumen de muestra recogida. Las muestras de sangre a través de las venas de la cola laterales también se puede realizar en el ratón como se describe aquí con algunas modificaciones menores. En primer lugar, se pueden usar sólo pequeñas de calibre (27 G) catéteres. En segundo lugar, se recomienda utilizar un tubo de contención, en lugar de una envoltura, para inmovilizar los ratones. El volumen de sangre que se puede obtener desde el ratón usando venopunción del haz vascular submandibular (200-500 l) es mayor que se pueden recoger de forma segura desde la vena de la cola (200 l máximo). Debido a que el muestreo de sangre del paquete vascular submandibular requiere restricción mínima y puede producir más sangre, ésta es la ruta preferida para el muestreo en el ratón. La rapidez con la que se puede realizar este procedimiento, junto con su naturaleza mínimamente invasiva, también minimiza la perturbación potencial de las medidas basadas en la sangre por la respuesta de estrés agudo 6. Losrespuesta al estrés agudo puede alterar los niveles circulantes de muchas moléculas, incluyendo las interleucinas y otros factores inmunes activa 7, hormonas del eje hipotálamo-hipófisis-suprarrenal 8, hormonas en el sistema nervioso simpático 9, 10 grelina, opioides endógenos 11, dopamina, y serotonina 12. Si se desean medidas de reposo circulantes de estas moléculas u otros regulados por estas moléculas, es importante para minimizar la respuesta al estrés, que se activa dentro de tan poco como un minuto del inicio de la exposición factor estresante. Las respuestas al estrés no sólo alteran la composición de la sangre, sino que también representan un obstáculo técnico para el muestreo debido a la constricción de los vasos de la sangre a través de una mayor unidad del sistema nervioso simpático. Se vuelve cada vez mayor dificultad para obtener el flujo de sangre constante de una rata que está montando una respuesta de estrés agudo. Por lo tanto, la angustia del animal debe ser minimzado con el fin de obtener rápidamente muestras que reflejan el estado fisiológico de interés.

Biomarcadores obtenidos a partir de sangre proporcionan utilidad de diagnóstico, predicción y estratificación medidas en muchos contextos clínicos, incluyendo la enfermedad cardiovascular 1, ciencias cáncer 2 y enfermedad psiquiátrica 3. También pueden ser utilizados en la ciencia básica para evaluar el &quot;estado&quot; de un organismo, incluyendo el grado de hambre, la inflamación, o el estrés presente. Estas medidas pueden ser influenciados por variables que pueden o no pueden ser críticos a la pregunta de interés, incluyendo la hora del día en que se obtiene la muestra y el sexo de los sujetos. También puede estar influenciada por la tensión inducida durante el propio procedimiento de muestreo de sangre. Las hormonas del estrés y la percepción del dolor pueden alterar rápidamente la composición de la sangre. Los roedores son el animal de laboratorio más comúnmente utilizado, y múltiples métodos han sido desarrollados para la recogida de sangre. El método ideal de muestreo de sangre debe tener physiologica mínimol impacto sobre el animal, no requieren anestesia, permite muestreo rápido y repetido, y proporcionar el volumen de sangre suficiente para numerosas aplicaciones posteriores. Técnicas populares para la recogida de la sangre, como la cateterización de la vena yugular o la amputación de la cola de punta no cumplen con estos criterios. El objetivo de este protocolo es describir una técnica de muestreo de sangre para su uso en ratas que es mínimamente estresante, no requiere anestesia, permite múltiples colecciones de sangre dentro de un mismo tema, y ​​ofrece un volumen de muestra relativamente grande tal que varios ensayos pueden realizarse en una sola muestra. El objetivo de este método es la obtención de muestras de sangre que están mínimamente influenciados por la respuesta de estrés agudo.

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	<tbody>
		<tr>
			<td>Sodium heparin (1000 USP units/ml)</td>
			<td>Patternson Veterinary Supply</td>
			<td>25021040010</td>
			<td></td>
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		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>JT Taylor</td>
			<td>JT2020-01</td>
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			<td>Dermachlor Rinse-Chlorhexadine</td>
			<td>Butler Schein</td>
			<td>6356</td>
			<td>Topical antiseptic solution, 2% chlorhexidine gluconate</td>
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			<td>SURFLO Winged Infusion Sets, Terumo, butterfly catheters</td>
			<td>VWR Scientific</td>
			<td>TESV25BLK</td>
			<td></td>
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			<td>BD Tuberculin 1cc syringes</td>
			<td>VWR Scientific</td>
			<td>BD309659</td>
			<td></td>
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			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>89202-682</td>
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			<td>500 &#956;l microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>21150-330</td>
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			<td>Scissors, stainless steel, 5&#34;</td>
			<td>VWR Scientific</td>
			<td>82027-586</td>
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			<td>500ml plastic beaker</td>
			<td>VWR Scientific</td>
			<td>414004-149</td>
			<td></td>
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		<tr>
			<td>Clean cloth wrap</td>
			<td>Butler Schein</td>
			<td>2993</td>
			<td></td>
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		<tr>
			<td>Velcro tape, .75&#34; width</td>
			<td>Monoprice</td>
			<td>B004AF9II6</td>
			<td>Hook and loop tape</td>
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		<tr>
			<td>Timer</td>
			<td>VWR Scientific</td>
			<td>62344-641</td>
			<td></td>
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sampling blood from the lateral tail vein of the rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Plasma de la sangre obtenida de la vena lateral de la cola como se describe en el protocolo da una muestra de plasma que era translúcida y de color amarillo pálido en apariencia. Como se muestra en la Figura 1, la hemólisis en una muestra imparte un tinte rojo a la plasma. La respuesta de estrés agudo puede alterar rápidamente la composición de la sangre. Por ejemplo, la concentración de corticosterona circulante puede aumentar notablemente dentro de 10 minutos de exposición factor de estrés, como se muestra en la Figura 2. Los bajos niveles basales de corticosterona obtenidos con este método antes de la exposición estresores revelan que el procedimiento de muestreo en sí no es una fuente significativa de estrés. <img alt="Figura 1" src="/files/ftp_upload/52766/52766fig1.jpg" /> Figura 1: se muestra el aspecto de la muestra (A) Una muestra hemolizada.. Después de la centrifugación, el plasma o suero capa (superficie indicada por la ar negrofila) aparece teñida de color rosa o rojo. Tintes más oscuros indican mayores niveles de hemólisis. (B) Después de la centrifugación, una muestra recogida correctamente tendrá un claro, aspecto amarillento a la banda superior (superficie indicada por la flecha negro), que corresponde a la del plasma no hemolizado o suero. Cuando la eliminación de esta capa, es importante para no molestar a la sangre entera subyacente, ya sea empujando la punta de la pipeta en toda la capa de sangre o por aspiración de algunos de toda la sangre en la punta. Cualquier plasma o suero contaminado con sangre entera deben desecharse. <img alt="Figura 2" src="/files/ftp_upload/52766/52766fig2.jpg" /> Figura 2: corticosterona plasma se eleva rápidamente después de una experiencia estresante Se obtuvo sangre de la vena lateral de la cola de ratas adultas Long-Evans hembra antes y 10 min tras la exposición a 4 tonos (10 seg, 2 kHz, 85 dB) co-terminación. con footshocks (1 seg, 350 mu). Corticosterona en plasma de sangre al inicio del estudio (290,4 ± 138,8 pg / ml) fue significativamente menor que los niveles observados 10 min después de la presentación de la tensión de descarga en las patas (2204,8 ± 454,5 pg / ml, p = 0,02, n = 4), según lo determinado por t pareada -test. *, P &lt;0,05

Watch this Scientific Journal Video about Sampling Blood from the Lateral Tail Vein of the Rat at JoVE.com

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Toma de muestras de sangre de la vena lateral de la cola de la rata

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

sampling-blood-from-the-lateral-tail-vein-of-the-rat

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JoVE Journal

Biology

102.1K vistas.  Massachusetts Institute of Technology. Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Video: Sampling Blood from the Lateral Tail Vein of the Rat

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

La extracción de sangre de las ratas a través de la vena safena y por punción cardiaca

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Investigación

Biología

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-disección de cerebro de rata para el ARN o la extracción de proteínas a partir de región específica del cerebro

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Enriquecimiento de las células NK a partir de sangre humana con el Kit de RosetteSep de Tecnologías de la StemCell

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Extracción de sangre del cangrejo herradura de América, Limulus polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Preparación de los tendones Cola de Rata de Estudios biomecánicos y Mechanobiological

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

La cuantificación individual Microvessel Permeabilidad en aislado de rata Blood-perfundido Preparación de pulmón

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

Técnicas de muestreo de sangre no terminal en los conejillos de Indias

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Eficiente iPS Generación Celular de sangre usando episomas y HDAC inhibidores

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Detectar el establecimiento de suministro de sangre compartido en ratones parabióticos mediante inyección de glucosa en vena caudal

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...