Agradecemos Virginia Doherty e Junmei Yao para a assistência técnica. Esta pesquisa foi financiada pelo NIMH (R01 MH084966) eo Escritório de Pesquisa do Exército dos EUA e da Agência de Projetos de Pesquisa Avançada de Defesa (conceder W911NF-10-1-0059) para KAG.

Todas as experiências foram realizadas utilizando ratos adultos machos Long-Evans. Todos os procedimentos estavam de acordo com os Institutos Nacionais de Saúde (NIH) Guia para o Cuidado e Uso de Animais de Laboratório e foram aprovados pelo Comitê de Cuidado e Uso de Animal Institucional do Instituto de Tecnologia de Massachusetts e do Animal Care e Use o Office Revisão da o USAMRMC. 1. Preparação <ol><li> Heparinise o cateter e seringa através da colocação da agulha blindado em um tubo de 500 ul contendo heparina (1.000 unidades USP / ml) e, em seguida, aspirar e expelir solução de heparina, através da agulha. <ol><li> Anexar um cateter de borboleta para a seringa. Manter a blindagem sobre a agulha do cateter para proteger a ponta afiada de danos. </li><li> Retirar um volume de heparina que é ligeiramente maior do que o volume de sangue que serão recolhidos. Retire a seringa e encha-o com o ar. </li><li> Volte a ligar a seringa ao cateter e usar oar para expelir o excesso de solução de heparina; garantir que apenas quantidades vestigiais permanecer na tubagem, agulha e seringa. </li><li> Coloque o cateter estéril, com a seringa ainda fixa, em uma superfície estéril. </li></ol></li><li> Garantir rapidamente o rato em um pano limpo garantindo que previsão e patas traseiras estão em uma posição confortável e respiração é irrestrita. <ol><li> Prenda o envoltório com gancho e laço de fita; assegurar que os genitais externos não são constringido. </li><li> Peça a um assistente gentil e firmemente refrear o rato (abdômen e base da cauda) sobre uma superfície de trabalho sólida com o rabo pendurado na borda do balcão. </li></ol></li></ol> 2. Amostragem de sangue <ol><li> Imergir a cauda em 42 ° C para a água de 40-50 seg para dilatar os vasos sanguíneos e secar a cauda com uma toalha de papel. Localizar a veia da cauda para ser sangrado (girar todo o corpo, com a cauda para evitar a torção da cauda). Nota: O aquecimento suficiente da cauda é crítica para a rápida collection de uma amostra de sangue. Se a vasculatura é constringido, a colocação adequada do cateter é difícil, e o fluxo sanguíneo é muito reduzido. Uma almofada de aquecimento pode ser usada como uma alternativa à imersão em água. </li><li> Identificar o ponto de amostragem. Nota: A artéria se encontra ao longo do aspecto médio-dorsal da cauda; não use isso para amostragem. <ol><li> Alvo ambos os lados esquerdo e direito veias da cauda que se encontram lateralmente à artéria. A pigmentação da cauda, ​​que varia de acordo com a tensão e aumenta com a idade, algumas podem obscurecer da vasculatura. Alvo de um porção da veia na porção inferior da cauda. </li><li> Limpe a área de alvo com solução anti-séptica de clorexidina 2%. </li></ol></li><li> Criar uma pressão negativa na seringa e um cateter, retirando o êmbolo de zero a cerca de 50 ul. </li><li> Segure a cauda gentil e firmemente perto da ponta para manter a cauda em linha reta para coleta de amostras. Certifique-se de que o fluxo sanguíneo não for obstruído por um aperto excessivamente apertado.</li><li> Lentamente inserir o cateter na veia num ângulo raso cerca de 5 cm a partir da ponta da cauda. Quando a veia é penetrado, o sangue irá fluir para dentro do cateter. Lentamente, retirar o êmbolo da seringa para recolher o volume desejado a uma taxa constante (~ 20 ul por segundo). <ol><li> Consulte o pessoal veterinário para obter informações sobre o volume de sangue máxima que pode ser cobrada. A quantidade máxima de sangue que devem ser recolhidos depende do status de peso e saúde do rato. Não retirar mais de 15% do volume total de sangue em um período de 14 dias. Nota: O sangue é muito mais difícil de recolher a partir de animais que foram agudamente estressados ​​nos minutos antes da coleta da amostra, pois os hormônios do estresse contraem a vasculatura. Por exemplo, mover gaiola do rato a um novo quarto, levando vários minutos para embrulhar o animal, ou inserção repetida do cateter em uma veia são todos susceptíveis de desencadear uma resposta de estresse agudo. </li><li> Facilitar bloofluxo d por &quot;ordenha&quot; a veia. Passar um dedo ao longo do comprimento da veia, a partir da base para a ponta da cauda, ​​mas permanecem mais de 2 cm a partir da ponta da agulha inserida ou o cateter pode ficar desalojado da veia. </li><li> Se o sangue não pode ser recolhido com êxito a partir do local inicial de penetração do cateter, re-inserir a agulha mais acima na veia. Se o sangue foi coletado no local inicial, re-pressurizar a agulha desligando e voltando a ligar o cateter e seringa antes da re-inserção na veia. Em geral, evitar penetrações adicionais. </li><li> Como várias penetrações pode causar o colapso da veia da cauda, ​​em que o fornecimento de sangue para a cauda é cortada eo tecido macio cauda torna-se necrosada, eutanásia do rato se houver colapso veia da cauda. </li></ol></li><li> Quando o volume da amostra adequada ser coletada, liberar a pressão na seringa por desconectar e reconectar o cateter. Aspirar usando ligeiramente o êmbolo da seringa (~ 50 &amp; #181; l), e retirar a agulha da veia. Nota: Se a agulha seja retirada sem primeiro libertar a pressão na seringa, o sangue vai escorrer a partir da agulha. </li><li> Resumidamente aplicar pressão ao local de inserção para parar o sangramento, e limpe a área com solução anti-séptica. Retorne o rato à sua gaiola. </li></ol> 3. Processar a amostra de sangue <ol><li> Ar aspirado para garantir que nenhum sangue permanece dentro da agulha do cateter, e usar uma tesoura para cortar o tubo de cateter logo acima da agulha. Expelir o sangue em um estéril 1,5 ml tubo de microcentrífuga. Nota: Se o sangue é empurrado através da agulha, a força de corte pode levar as células vermelhas do sangue para romper o que pode interferir com muitos ensaios a jusante. Remover a agulha, para evitar hemólise. <ol><li> Para recolher o plasma sanguíneo, que contêm tubos de uso EDTA como anticoagulante (aqui, utilizar 10 ul de EDTA 0,1 M para 200-400 ul de sangue; garantir a concentração de EDTA utilizada não interfere wom o ensaio downstream) e colocar no gelo. <ol><li> Rotação amostras de sangue total a 2100 xg numa centrifugadora refrigerada (4 ° C) durante 10 min até 10 min de colheita. Elui-se o plasma, evitando perturbar as camadas de células de sangue vermelhas e brancas. </li></ol></li><li> Para a coleta de soro do sangue, amostras de lugar (sem anticoagulante) à temperatura ambiente por até 30 min para permitir a coagulação. Girar os tubos de recolha numa centrifugadora refrigerada (4 ° C) a 2000 x g. O soro pode então ser eluída. </li></ol></li><li> Utilize amostras imediatamente, ou armazenar a -80 ° C por até um ano. </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>

Os autores não têm nada a revelar.

Aqui, nós descrevemos um processo rápido e simples para a obtenção de uma amostra de sangue de um rato que oferece vantagens significativas sobre outras técnicas vulgarmente utilizadas. Em primeiro lugar, ele não necessita de anestesia, em contraste com a amostra a partir da veia jugular ou da cavidade retro-orbital. Quando as amostras de sangue são recolhidos em torno procedimentos comportamentais, a administração de anestésicos é indesejável porque pode interferir com a aprendizagem e memória 4,5. Em segundo lugar, ele oferece a capacidade de recolher volumes de sangue maiores do que outras técnicas de punção venosa, como a recolha das veias safenas pedal ou dorsais. Utilizando a técnica descrita aqui, até 1,5 ml de sangue pode ser coletado de um rato em um único ponto do tempo, um volume que facilmente permite que vários ensaios para ser executado em paralelo. Finalmente, este procedimento minimiza o potencial de dano tecidual comparado a amputação da ponta da cauda ou sangramento retro-orbital. A utilização deste procedimento facilita o cumprimento com o animal Welfare Act eo Guia para o Cuidado e Uso de Animais de Laboratório, que exigem minimizar a dor eo sofrimento que resulta de procedimentos de laboratório realizados em animais. Recomenda-se que os investigadores novas para praticar este método e as técnicas de retenção de sangramento da cauda, ​​a fim de minimizar o tempo que os animais experimentais são contidos. O sangue coletado em menos de 3 minutos do início de contenção proporciona ótimos resultados. O protocolo aqui descrito pode ser utilizado para a amostragem de 1 a 4 vezes por semana, mas não mais do que duas vezes por dia. Enquanto colecções repetidas de sangue pode ser realizado, diferentes pontos de amostragem que se deslocam para cima a partir da base da cauda deve ser usado, e as veias da cauda esquerda e direita deve ser alternado como locais de amostragem. O volume total de sangue de roedores é 6-7% do seu peso corporal, e não mais do que 15% do volume total de sangue deve ser recolhido dentro de um período de 2 semanas. Soroou plasma compreende cerca de 40-60% do volume da amostra recolhida. A amostragem de sangue através das veias da cauda laterais podem também ser executada no rato, como descrito aqui com algumas pequenas modificações. Em primeiro lugar, pode ser utilizado apenas um pequeno calibre (27 L) cateteres. Em segundo lugar, é recomendado o uso de um limitador tubo, em vez de um envoltório, para imobilizar os ratinhos. O volume de sangue que pode ser obtida a partir do ratinho usando punção venosa do feixe vascular submandibular (200-500 uL) é maior do que pode ser recolhida de forma segura a partir da veia da cauda (200 ul máxima). Porque a amostragem de sangue do feixe vascular submandibular requer contenção mínima e pode produzir mais sangue, esta é a via preferida para a amostragem no mouse. A rapidez com que este procedimento pode ser realizado, juntamente com a sua natureza minimamente invasiva, também minimiza a perturbação potencial de medidas baseadas em sangue pela resposta aguda de estresse 6. Oresposta ao estresse agudo pode alterar os níveis circulantes de muitas moléculas, incluindo interleucinas e outros fatores imunológicos-ativo 7, hormônios do eixo hipotálamo-hipófise-adrenal, 8 hormônios no sistema nervoso simpático 9, 10 grelina, opióides endógenos 11, dopamina e serotonina 12. Se são desejados repouso circulante medidas dessas moléculas ou outros reguladas por estas moléculas, é importante para minimizar a resposta ao stress, que é accionado dentro de tão pouco como um minuto do início da exposição ao stressor. As respostas ao estresse não só alterar a composição do sangue, mas também representam um obstáculo técnico para coleta de sangue por causa da constrição dos vasos através de uma maior unidade do sistema nervoso simpático. Torna-se cada vez mais difícil obter o fluxo de sangue constante de um rato que está montando uma resposta ao estresse agudo. Por isso, a angústia do animal deve ser mínimazada, a fim de se obter rapidamente as amostras que reflectem o estado fisiológico de interesse.

Biomarkers obtidos a partir de sangue fornecer informações úteis de diagnóstico, previsão, e estratificando medidas em muitos contextos clínicos, incluindo a doença cardiovascular 1, 2 ciências câncer e doença psiquiátrica 3. Eles também podem ser usados ​​em ciência básica para avaliar o &quot;estado&quot; de um organismo, incluindo o grau de fome, a inflamação, ou o stress presente. Tais medidas podem ser influenciadas por variáveis ​​que podem ou não podem ser críticos para a questão dos juros, incluindo o tempo do dia em que a amostra é obtida eo gênero dos indivíduos. Ele também pode ser influenciada pela tensão induzida durante os procedimentos de amostragem de sangue em si. Hormonas de stress e a percepção de dor pode alterar-se rapidamente a composição do sangue. Os roedores são o animal de laboratório mais utilizada, e vários métodos foram desenvolvidos para a recolha de sangue. O método ideal de coleta de sangue deve ter Physiologica mínimol impacto sobre o animal, não necessitam de anestesia, permitir a amostragem rápida e repetida, e fornecer o volume de sangue suficiente para inúmeras aplicações a jusante. Técnicas populares para recolha de sangue, como cateterização da jugular amputação da ponta da cauda veia ou não atender a esses critérios. O objectivo deste protocolo é descrever uma técnica de coleta de sangue para uso em ratos que é minimamente estressante, não requer anestesia, permite múltiplas coletas de sangue dentro de um único assunto, e fornece um volume relativamente grande amostra de tal forma que vários ensaios podem ser realizados numa única amostra. O objectivo deste método é a obtenção de amostras de sangue que são minimamente influenciado pela resposta de stress 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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			<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>
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			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>89202-682</td>
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		<tr>
			<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>
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			<td>Clean cloth wrap</td>
			<td>Butler Schein</td>
			<td>2993</td>
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			<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.

O plasma sanguíneo recolhido a partir da veia lateral da cauda, ​​tal como descrito no protocolo dá uma amostra de plasma que era translúcida e de cor amarela pálida na aparência. Como mostrado na Figura 1, numa amostra de hemólise confere uma tonalidade vermelha com o plasma. A resposta ao estresse agudo pode alterar rapidamente a composição do sangue. Por exemplo, circulando concentração de corticosterona pode aumentar significativamente dentro de 10 minutos de exposição estressor, como mostrado na Figura 2. Os baixos níveis de corticosterona basais obtidos com este método, antes da exposição a factores de stress revelam que o próprio procedimento de amostragem não é uma fonte importante de stress. <img alt="Figura 1" src="/files/ftp_upload/52766/52766fig1.jpg" /> Figura 1:. Aparência da amostra (A) Uma amostra hemolisada é mostrado. Após centrifugação, o plasma ou soro camada (superfície indicada pela AR pretolinha) parece tingido de rosa ou vermelho. Matizes mais escuras indicam maiores níveis de hemólise. (B) Depois de centrifugação, uma amostra adequadamente recolhido terá um aspecto límpido, amarelado a banda superior (superfície indicada pela seta preta), que corresponde ao plasma ou soro não hemolisado. Quando a remoção desta camada, é importante para não perturbar o sangue total subjacente, quer empurrando a ponta da pipeta em toda a camada de sangue ou por aspiração de alguns do sangue total para a ponta. Qualquer plasma ou soro contaminado com sangue total deve ser descartado. <img alt="Figura 2" src="/files/ftp_upload/52766/52766fig2.jpg" /> Figura 2: corticosterona do plasma é rapidamente elevada após uma experiência estressante Foi obtido sangue a partir da veia lateral da cauda de ratos do sexo feminino Long-Evans adultos antes e 10 min após a exposição a 4 tons (10 sec, 2 kHz, 85 dB) co-terminal. com footshocks (1 seg, 350 mA). Corticosterona no plasma de sangue na linha de base (290,4 ± 138,8 pg / ml) foi significativamente menos do que os níveis observados 10 minutos a seguir à apresentação da tensão de choque nas patas (2204,8 ± 454,5 pg / ml, p = 0,02, n = 4), tal como determinado por t emparelhado -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.

A amostragem de sangue a partir da veia lateral da cauda do rato

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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Biology

102.1K visualizações.  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.

Vídeo: 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.

Desenho de sangue de ratos através da veia safena e por punção cardíaca

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: ...

Biologia

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-dissecção de cérebro de um rato para RNA ou Extração de Proteína da região específica do cérebro

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.

Enriquecimento das células NK do sangue humano com o Kit RosetteSep de 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 ...

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.

Coleta de sangue do caranguejo em ferradura americano, 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.

Preparação de tendões da cauda de rato de Estudos biomecânicos e 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.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

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.

Quantificar Único microvasos Permeabilidade em perfusão isolada-Sangue Rat Lung Preparação

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 coleta de sangue não terminal em cobaias

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 Geração células iPS a partir de sangue, utilizando epissomas e HDAC Inibidores

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.

Detectando estabelecimento de suprimento de sangue compartilhado em camundongos parabióticos por injeção de glicose em veia caudal

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