Os autores gostariam de agradecer as Dras. Paul Huang e Dmitriy Atochin para uso de DMT pressão myograph e as Dras. Binglan Yu e Chong Lei para a prestação de ratos alimentados com dieta rica em gordura ou dieta padrão. Fonte de financiamento Este trabalho foi apoiado pelo cientista Desenvolvimento Grant 10SDG2610313 da American Heart Association (a ES Buys) e um Miles Shore 50 programa Fellowship Anniversary Eleanor e for Scholars em Medicina pela Harvard Medical School (a ES Buys).

1. Preparação da Solução <ol><li> Estoque 500X EDTA: pesar 500 mg EDTA-Na 2 • 2H 2 O e dissolver em 50 ml de água deionizada. Guarde à temperatura ambiente. </li><li> KCl solução despolarizante. <ol><li> Preparar a solução estoque K10X: 3,69 g NaCl, 18,64 g de KCl, 0,36 g MgSO4 anidro, 0,41 g KH 2 PO 4, 0,46 g de CaCl2 • 2H 2 O. Dissolve-se em 250 ml de água desionizada. Guarde à temperatura ambiente. </li><li> Para 100 ml de solução final de KCl 1X despolarizante: Dissolver 0,21 NaHCO3, 0,18 g de glicose em 90 ml de água desionizada. Adicionar 10 ml da solução de K10X. Adicionar 95 ul de solução de EDTA 500X. Misturar bem. Armazenar a 4 ° C. É dentro de uma semana. </li></ol></li><li> Preparar solução fresca de HEPES-PSS no dia da experiência (para 1 litro). <ol><li> 6,96 g de NaCl, 0,35 g de KCl, 0,288 g de MgSO4 • 7H 2 O, 0,1605 g de KH 2 PO 4, 2,38 g de HEPES. Dissolve-se em 100 mlde água desionizada. Marcar como frasco A. </li><li> 0,312 g CaCl 2 • 2H 2 O. Dissolve-se em 100 ml de água desionizada. Marcar como frasco B. </li><li> 1,25 g de NaHCO3, 0,991 g de glicose. Dissolve-se em 98 ml de água desionizada. Marcar como frasco C. </li></ol></li><li> Adicionam-se 2 ml de EDTA num frasco de 500X C. Aqui 700 ml de água desionizada num balão de grande. Deite o conteúdo do balão A, B e C em grande balão com vórtex contínuo. Ajustar o pH para 7,4 com NaOH. </li></ol> 2. Drogas usadas <ol><li> A fenilefrina (10 -2 M): Dissolve-se 20,37 mg em 10 ml de água desionizada. Alíquota de 1 ml e armazenar a -80 ° C. Diluir em série para obtenção de 10 -3, 10 -4, 10 -5 e 10 -6 mol / L no dia da experiência. </li><li> A acetilcolina (10 -2 M): Dissolve-se 18,17 mg em 10 ml de água desionizada. Alíquota de 1 ml e armazenar a -80 ° C. Diluir em série para obtenção de 10 -3, 10 -4, 10 -5, -6 e 10mol / l no dia da experiência. </li></ol> 3. Ratos Macho WT e sGCα 1 - / - murganhos no fundo S6 e B6 foram estudados ao longo deste estudo. O sGCα 1 - / - foram gerados como descrito anteriormente 4,5. Habitação e procedimentos que envolvem animais de laboratório (ratos) foram aprovados pela Subcomissão de Pesquisa Animal Care do Massachusetts General Hospital, Boston, MA. 4. Medição de reatividade vascular em artéria mesentérica <ol><li> Eutanásia dos ratinhos com pentobarbital (200 mg / kg, ip). Abra a cavidade abdominal e dissecar o tecido mesentérica. Isolar e dissecar uma artéria mesentérica de 3 ª ordem isenta de tecido conjuntivo e adiposo e colocar a artéria no gelado Hepes PSS solução pré-equilibrada com 95% de O2 / 5% de CO 2 durante 15 min. Cortar anéis de 2-3 mm de comprimento, sem ramificação do mesentéricaartéria. A diferenciação entre artérias e veias nível micro vasos é problemático quando não há sangue nos vasos. Portanto, recomendamos identificar a embarcação enquanto ela ainda está intacta no animal antes de dissecá-lo para fora. </li><li> Preencher as cânulas de vidro na câmara de pressão myograph (DMT Modelo 110P &amp; 11P versão 1.31, Figura 2) com a solução de HEPES-PSS com a ajuda de uma seringa de 10 ml por meio de válvulas de entrada e de saída. Preenchimento de cânula deve ser feito com cuidado e com cuidado, pois a pressão excessiva pode danificar o transdutor frágil ligado a cânulas. Após o preenchimento cânulas fechar tanto de entrada e válvulas de saída com força. </li><li> Montar uma extremidade do navio para cânula direito e amarrá-lo com cuidado e com um fio fino de fio de náilon. Com a ajuda de uma seringa, lave e preencher o recipiente com solução HEPES através da válvula de admissão. Trazer a cânula esquerda para mais perto do navio e montado na outra extremidade do recipiente sobre ele. Amarre-o com fio de náilon. Preenchera câmara com até 10 ml com a solução de HEPES. Entrada para o vazamento empurrando suavemente solução HEPES, através da válvula de entrada com a ajuda de uma seringa. </li><li> Instalar a câmara na myograph e sob a câmara de video. Inicie o oxigénio e calor (37 ° C) na câmara. Ligação a válvula de admissão com o tubo de P1 a partir do primeiro reservatório para a solução de HEPES, enquanto a válvula é fechada. Que qualquer bolha e solução passe através do tubo até que não existam bolhas no tubo. Abrir a válvula. </li><li> Ligação a válvula esquerdo da câmara com o tubo P2 proveniente do regulador de pressão. Novamente verifique se há bolhas. Não deve haver quaisquer bolhas ou vazamento em todo o sistema. </li><li> Vá para o menu pressão sobre o painel de mio-interface ou no software e ligar a bomba enquanto desligar o fluxo. </li><li> Abra o programa e clique em Collect. Veículo deve ser visto no ecrã agora (Figura 3). O vaso é visualizado com uma câmara montada num microscópio, permitindo monitoring e análise do lúmen do vaso, o diâmetro do vaso, e a espessura da parede. </li><li> Aumentar gradualmente a pressão interluminal através da válvula P1 seleccionando pressão P1 no painel de mio-interface ou no programa e insira o valor da pressão da seguinte forma; 5 mmHg → 10 → 20 → 40 → 60 mmHg. Veículo algures tende a torcer ou convolute enquanto a pressão está a aumentar, ajustar a tensão ou tensão de acordo com a ajuda de microposicionador vertical ou longitudinal (Figura 2). Equilibrar a embarcação a 60 mmHg e 37 ° C, pelo menos durante 45 min. Altere a solução do banho uma vez com HEPES pré-aquecido durante equilíbrio. </li><li> Aplicar 10 ml de solução despolarizante de KCl, pré-aquecida a 37 ° C, para o banho de despolarizar totalmente as células musculares lisas e alcançar a constrição máxima. Depois de constrição estável, lavar a banheira com solução HEPES 3 vezes a cada 10 min. </li><li> Verificar a viabilidade do vaso e da integridade do endotélio, por estabilidade e reprodutibilidaderespostas comestíveis à adição de fenilefrina (10 -5 M) e acetilcolina (10 -5 M). Lavar 3 vezes com solução tampão HEPES a cada 10 min. </li><li> Adicionar fenilefrina (10 -5 M) para preconstrict do navio, e quando uma constrição estável foi obtida, efectuar uma curva de resposta de concentração-cumulativa vasodilatação (CDC), por adição sequencial de doses crescentes de acetilcolina (10 -9, 3 x 10 - 9, 10 -8, 3 x 10 -8, 10 -7, 3 x 10 -7, 10 -6, 3 x 10 -6 e 10 -5 M). Após a última dose, lavar 3 vezes com solução HEPES a cada 10 minutos antes de iniciar novo CRC. </li><li> Determinar o diâmetro do lúmen passiva através da aplicação de Ca 2 + PSS e contendo 2 mM de EGTA. A partir do diâmetro luminal medida traçados gravado para cada resposta a dose (Figura 4), ​​que será utilizado para calcular todos os parâmetros. </li><li> Expresse todas as respostas de relaxamento acetilcolina como percentagem mudança no diâmetro do lúmen, após a pré-fenilefrina em comparação com a diferença entre o diâmetro livre de cálcio e diâmetro após constrição fenilefrina, usando a seguinte equação: </li><li> Dilatação percentual = 100% x [(D x - D i) / (D-Ca livre - D i)], em que D é o diâmetro do lúmen medido e x, i e Ca-livre denotar diâmetros arteriais para cada dose de agonista (x), segue diâmetro inicial fenilefrina constrição (i), e em Ca 2 + tampão livre (Ca-free). </li></ol> 5. Análise Estatística Todas as medições contínuas são expressos como média ± SEM. A reatividade vascular em artérias mesentéricas é analisada por medidas repetidas ANOVA de duas vias. Em todos os casos, P &lt;0,05 é considerado estatisticamente significativo.

<ol>
	<li>Bridges, L. E., Williams, C. L., Pointer, M. A., Awumey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+artery+contraction+and+relaxation+studies+using+automated+wire+myography.">Mesenteric artery contraction and relaxation studies using automated wire myography.</a> J. Vis .Exp. (55), e3119 (2011).</li><li>Arribas, S. M., Daly, C. J., McGrath, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+vascular+remodeling+by+confocal+microscopy.">Measurements of vascular remodeling by confocal microscopy.</a> Methods Enzymol. 307, 246-273 (1999).</li><li>Lei, C., Yu, B., 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=Inhaled+Nitric+Oxide+Attenuates+the+Adverse+Effects+of+Transfusing+Stored+Syngeneic+Erythrocytes+in+Mice+with+Endothelial+Dysfunction+after+Hemorrhagic+Shock.">Inhaled Nitric Oxide Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock.</a> Anesthesiology. , (2012).</li><li>Buys, E. S., Cauwels, A., 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=sGC&#945;1&#946;1+attenuates+cardiac+dysfunction+and+mortality+in+murine+inflammatory+shock+models.">sGC&#945;1&#946;1 attenuates cardiac dysfunction and mortality in murine inflammatory shock models.</a> Am. J. Physiol. Heart Circ. Physiol. 297 (2), H654-H663 (2009).</li><li>Buys, E. S., Sips, 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=Gender-specific+hypertension+and+responsiveness+to+nitric+oxide+in+sGC&#945;1+knockout+mice.">Gender-specific hypertension and responsiveness to nitric oxide in sGC&#945;1 knockout mice.</a> Cardiovasc. Res. 79 (1), 179-186 (2008).</li><li>Panza, J. A., Quyyumi, A. A., Brush, J. E., Epstein, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+endothelium-dependent+vascular+relaxation+in+patients+with+essential+hypertension.">Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension.</a> N. Engl. J. Med. 323 (1), 22-27 (1990).</li><li>Huang, P. L., Huang, Z., 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=Hypertension+in+mice+lacking+the+gene+for+endothelial+nitric+oxide+synthase.">Hypertension in mice lacking the gene for endothelial nitric oxide synthase.</a> Nature. 377 (6546), 239-242 (1995).</li><li>Friebe, A., Koesling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+NO-sensitive+guanylyl+cyclase:+what+we+can+learn+from+genetic+mouse+models.">The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models.</a> Nitric Oxide. 21 (3-4), 149-156 (2009).</li><li>Ehret, G. B., Munroe, P. B., 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=Genetic+variants+in+novel+pathways+influence+blood+pressure+and+cardiovascular+disease+risk.">Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.</a> Nature. 478 (7367), 103-109 (2011).</li><li>Buys, E. S., Raher, M. J., 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=Genetic+modifiers+of+hypertension+in+soluble+guanylate+cyclase+alpha1-deficient+mice.">Genetic modifiers of hypertension in soluble guanylate cyclase alpha1-deficient mice.</a> J. Clin. Invest. 122 (6), 2316-2325 (2012).</li><li>Kauffenstein, G., Laher, I., Matrougui, K., Guerineau, N. C., Henrion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+G+protein-coupled+receptors+in+microvascular+myogenic+tone.">Emerging role of G protein-coupled receptors in microvascular myogenic tone.</a> Cardiovascular Research. 95 (2), 223-232 (2012).</li><li>Ruilope, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension+in+2010:+Blood+pressure+and+the+kidney.">Hypertension in 2010: Blood pressure and the kidney.</a> Nat. Rev. Nephrol. 7 (2), 73-74 (2011).</li><li>Michael, S. K., Surks, H. 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=High+blood+pressure+arising+from+a+defect+in+vascular+function.">High blood pressure arising from a defect in vascular function.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (18), 6702-6707 (2008).</li><li>Mendelsohn, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+hypertension,+the+kidney+is+not+always+the+heart+of+the+matter.">In hypertension, the kidney is not always the heart of the matter.</a> J. Clin. Invest. 115 (4), 840-844 (2005).</li></ol>

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Camundongos são o modelo experimental de escolha para muitos pesquisadores, em parte por causa da possibilidade de introduzir modificações genéticas, gerando modelos de mouse para fisiopatologia humana. O estado vasoativas de pequena resistência, mas não dos vasos maiores conduíte define em grande parte a regulação do fluxo de sangue em todo o sistema vascular 11. O tamanho das artérias de resistência em animais pequenos, tais como ratos, previne a utilização de um fio myograph para estudar a fun...

Myograph sistemas de pressão são usadas para medir a função e as propriedades fisiológicas das pequenas artérias, veias e outros recipientes. Um pequeno segmento de uma artéria ou veia intactas é montada sobre dois pequenos cânulas de vidro e pressurizado a uma pressão luminal adequada, permitindo que o recipiente para manter a maior parte do seu características in vivo (Figuras 1 e 2). A condição fisiológica perto num myograph pressão reflecte o comportamento in vivo do leito vascular, o que permite a investigação das propriedades intrínsecas (por exemplo, o tom miogénica) de vasos isolados. Algumas das vantagens da Miografia Miografia pressão ao longo do fio, onde a contração muscular é avaliada através de acoplamento mecânico directo a um transdutor de força 1, incluem: (i) que as micro artérias de resistência, que definem a resistência global desenvolvido no sistema vascular, podem ser estudadas, enquanto myograph fio está limitado a maiores artérias condutas, (ii) tchapéu o risco de danificar o endotélio é reduzido uma vez que não há necessidade de fios de ser passado através do lúmen do vaso, (iii) que a forma natural do vaso é melhor mantida, e (iv) que dimensão navio pode ser estudado em uma ampla gama de pressões ou estresse de cisalhamento 2. Estudar micro vasos pode ser mais informativo do que estudar maiores artérias do conduíte para ajudar a compreender os mecanismos fisiopatológicos e moleculares que contribuem para a alteração do tônus ​​vascular em doenças cardiovasculares, como hipertensão. Por exemplo, a deficiência da função endotelial associada com a alimentação de ratos uma dieta rica em gorduras durante 8 semanas podia ser demonstrado em 2 ª ordem artérias mesentéricas 3, mas não em anéis da aorta (Figura 3). Outra vantagem é que a pressão Miografia constrição miogénica intrínseca do recipiente pressurizado está presente, e que o papel e a função do endotélio para este fenómeno pode ser estudada. Aqui, nós describe o uso de uma pressão myograph para estudar a reactividade vascular mesentérica de rato 3 ª ordem artérias resistência numa configuração de óxido nítrico prejudicada (NO)-cGMP sinalização.

<table border="1">
		<tbody>
		 <tr>
			<td>Name</td>
			<td>Company</td>
			<td>Cataloge No.</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
		 </tr>
		 <tr>
			<td>CaCl2 (2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
		 </tr>
		 <tr>
			<td>MgSO4 </td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
		 </tr>
		 <tr>
			<td>KH2PO4 </td>
			<td>Sigma</td>
			<td>P3786</td>
		 </tr>
		 <tr>
			<td>NaHCO3 </td>
			<td>Fisher Scientific</td>
			<td>BP328</td>
		 </tr>
		 <tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318</td>
		 </tr>
		 <tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP121</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Acros Organics</td>
			<td>AC20724</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma</td>
			<td>A6625</td>
		 </tr>
		 <tr>
			<td>Pressure Myograph System</td>
			<td>DMT</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

assessing murine resistance artery function using pressure myography

Sistemas myograph pressão são primorosamente útil na avaliação funcional das pequenas artérias, pressurizado a uma pressão transmural adequado. A condição fisiológica perto alcançado em Miografia pressão permite a caracterização aprofundada das respostas intrínsecas a estímulos farmacológicos e fisiológicos, os quais podem ser extrapolados para o comportamento in vivo do leito vascular. Myograph pressão possui diversas vantagens em relação myographs fios convencionais. Por exemplo, os vasos de resistência menores pode ser estudada a pressões intraluminais estritamente controlados e fisiologicamente relevante. Aqui, vamos estudar a possibilidade de 3 ª ordem artéria mesentérica (3-4 mm de comprimento), preconstricted com fenilefrina, a vaso-relaxamento em resposta à acetilcolina. Artérias mesentéricas são montados em duas cânulas ligadas ao sistema pressurizado, selado e que é mantida a uma pressão constante de 60 mmHg. O diâmetro do lúmen e o exterior do vaso são Continuously gravado com uma câmara de vídeo, que permite a quantificação da vasoconstrição e relaxação de vasos em resposta a fenilefrina e acetilcolina em tempo real, respectivamente. Para demonstrar a aplicabilidade do Miografia pressão para estudar a etiologia da doença cardiovascular, avaliou-se a função vascular endotélio-dependente em um modelo murino de hipertensão sistémica. Camundongos deficientes na subunidade α 1 da guanilato ciclase solúvel (sGCα 1 - / -) são hipertensos quando em um (S6) Fundo 129S6 (sGCα 1 -/-S6), mas não quando em um C57BL / 6 (B6) fundo (sGCα 1 -/-B6). Usando Miografia pressão, demonstramos que sGCα 1 deficiência resulta em alterações na vasorelaxation endotélio-dependente. A disfunção vascular é mais acentuada em sGCα 1 -/-S6 que em sGCα 1 -/-B6 ratos, provavelmente CONTRIBUTOng a maior pressão no sGCα 1 -/-S6 que em sGCα um -/-B6 ratinhos. Miografia pressão é uma técnica relativamente simples, mas sensível e mecanisticamente útil que pode ser utilizado para avaliar o efeito de diferentes estímulos na contracção e relaxamento vascular, aumentando, assim, o nosso conhecimento sobre os mecanismos subjacentes à doença cardiovascular.

NO está envolvido centralmente na manutenção da homeostase da pressão sanguínea tanto em humanos como 6 e 7, em modelos animais. A capacidade do NO para controlar o relaxamento do músculo liso vascular é mediada por guanilato ciclase solúvel (sGC), um enzima heterodimérica contendo heme que gera cGMP 8. Recentemente, uma pressão sanguínea de modificação variante genética foi identificada num local que contém a sGCα 1 e genes sGCβ 1, ilustrando a rel...

Watch this Scientific Journal Video about Assessing Murine Resistance Artery Function Using Pressure Myography at JoVE.com

Assessing Murine Resistance Artery Function Using Pressure Myography

Em Miografia pressão, um pequeno segmento de um vaso intacto é montado sobre duas pequenas cânulas e pressurizado a uma pressão adequada luminal. Aqui, descrevemos o método para medir a resposta vasorelaxation do mouse 3<sup&gt; Rd</sup&gt; Ordem artérias mesentéricas em C57 e sGCα<sub&gt; 1</sub<sup&gt; - / -</sup&gt; Ratos usando Miografia pressão.

Avaliando Murino Resistência Função Usando Miografia Pressão Arterial

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

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21.9K visualizações.  Massachusetts General Hospital, Harvard Medical School. Em Miografia pressão, um pequeno segmento de um vaso intacto é montado sobre duas pequenas cânulas e pressurizado a uma pressão adequada luminal. Aqui, descrevemos o método para medir a resposta vasorelaxation do mouse 3 Rd Ordem artérias mesentéricas em C57 e sGCα 1

Vídeo: Assessing Murine Resistance Artery Function Using Pressure Myography

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

Medição da pressão arterial em ratos usando Recording Volume de pressão, um método de cauda-manguito

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Biologia

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Bioensaios de Resistência a Inseticidas Monitoramento

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Explorando Arterial Músculo Liso KV7 função dos canais de potássio utilizando electrofisiologia patch clamp e Miografia Pressão

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secundária Previsão Estrutura Usando SHAPE de alta capacidade

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; Effective dampening of flow against pressure changes occurs over periods as short as ~15 sec and becomes progressively greater over longer time periods. Thus, slower changes in blood pressure are effectively blunted and faster changes or fluctuations pass through to cerebral blood flow relatively unaffected. The primary difficulty in characterizing the frequency dependence of cerebral autoregulation is the lack of prominent spontaneous fluctuations in arterial pressure around the frequencies of interest (less than ~0.07 Hz or ~15 sec). Oscillatory lower body negative pressure (OLBNP) can be employed to generate oscillations in central venous return that result in arterial pressure fluctuations at the frequency of OLBNP. Moreover, Projection Pursuit Regression (PPR) provides a nonparametric method to characterize nonlinear relations inherent in the system without a priori assumptions and reveals the characteristic non-linearity of cerebral autoregulation. OLBNP generates larger fluctuations in arterial pressure as the frequency of negative pressure oscillations become slower; however, fluctuations in cerebral blood flow become progressively lesser. Hence, the PPR shows an increasingly more prominent autoregulatory region at OLBNP frequencies of 0.05 Hz and below (20 sec cycles). The goal of this approach it to allow laboratory-based determination of the characteristic nonlinear relationship between pressure and cerebral flow and could provide unique insight to integrated cerebrovascular control as well as to physiological alterations underlying impaired cerebral autoregulation (e.g., after traumatic brain injury, stroke, etc.).

Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.

Avaliando Cerebral A autorregulação via Oscillatory Lower Body pressão negativa e Projeção Perseguição Regressão

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Medir a função respiratória em ratos usando Unrestrained pletismografia de corpo inteiro

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiac Pressão-Volume Análise loop usando Conductance Cateteres em Ratos

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Gravação de Intracavernosal pressão para avaliar a função erétil em roedores

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

Indução de insuficiência ventricular direita por constrição da artéria pulmonar e avaliação da função ventricular direita em camundongos

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and disease. EIM is obtained by applying a low-intensity, directionally focused, electrical current to a muscle of interest across a range of frequencies (i.e., from 1 kHz to 10 MHz) and recording the resulting voltages. From these, several standard impedance components, including the reactance, resistance, and phase, are obtained. When performing ex vivo measurements on excised muscle, the inherent passive electrical properties of the tissue, namely the conductivity and relative permittivity, can also be calculated. EIM has been used extensively in animals and humans to diagnose and track muscle alterations in a variety of diseases, in relation to simple disuse atrophy, or as a measure of therapeutic intervention. Clinically, EIM offers the potential to track disease progression over time and to assess the impact of therapeutic interventions, thus offering the opportunity to shorten the clinical trial duration and reduce sample size requirements. Because it can be performed noninvasively or minimally invasively in living animal models as well as humans, EIM offers the potential to serve as a novel translational tool enabling both preclinical and clinical development. This article provides step-by-step instructions on how to perform in vivo and ex vivo EIM measurements in mice and rats, including approaches to adapt the techniques to specific conditions, such as for use in pups or obese animals.

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals, pups).

Realização de Miografia de Impedância Elétrica In Vivo e Ex Vivo em Roedores

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...