Este trabalho é apoiado pela American Heart Association 14FTF20370058 (DMA) e NIH T32 HL007101-35 (DMA).

1. Preparações Conductance cateter e calibração de pressão <ol><li> Conectar cateter de condutância para o módulo de cateter hemodinâmica. Eletronicamente calibrar as medições de pressão e volume gravando pressão predefinida eo volume ajustado no módulo cateter. Grave um traçado de 0 mm Hg e 25 mm Hg (Figura 1A) e atribuir as voltagens para ambos os traçados de pressão (Figura 1B e 1C). Da mesma forma, gravar um rastreamento volume de 5 RVU e 25 RVU (Figura 1D) e atribuir as voltagens para ambos os traçados unidades (1E e 1F). </li><li> Confirme a calibração de pressão eletrônico com uma calibração de pressão manual, utilizando uma coluna de mercúrio. Montar a porta esfigmomanômetro manguito com uma torneira de 3 vias. Preencha um sistema de válvula hemostática de 3 portas, muitas vezes utilizados para angioplastia coronária, com água RT usando a porta lateral. <ol><li> Colocar a ponta de um cateter de condutância na válvula de hemostase cheia de fluido e garantir suavemente without torcer cateter. Ligue a válvula hemostática para o esfigmomanômetro e inflar a 200 mmHg e bloquear torneira de 3 vias. Examinar se a pressão medida corresponde com a pressão insuflado na esfigmomanômetro. </li></ol></li><li> Local cateter em soro fisiológico aquecido a 37 ° C, que é ao nível do campo operatório e pressão medida. Ajuste o controle de pressão até que as pressões registradas está em zero. </li></ol> 2. Anestesia / intubação <ol><li> Administrar cetamina / xilazina (80-100 / 10 mg kg -1) como uma injecção intraperitoneal. Nota: Os agentes anestésicos alternativos podem ser usados. Uma extensa lista de anestésicos são fornecidos em comentários anteriores desta técnica 11,12. Anestesiados apropriada pode ser confirmada por aperto suave da cauda. </li><li> Uma vez anestesiados, fazer a barba do pescoço e peito com máquinas de cortar cabelo e coloque em uma almofada aquecida. Manter a temperatura rectal do rato em 36,5-37,5 ºC. Baixas temperaturas corporaisirá resultar em taxas de coração deprimidos. Aplicar pomada para os olhos para prevenir a secura </li><li> Adicione uma incisão na linha média do pescoço e dissecar os músculos traqueais para expor a traqueia. Coloque um tubo endotraqueal através da boca, enquanto visualiza a traquéia para garantir a intubação, e conectar-se ao respirador. </li><li> Manter rato no ventilador durante o procedimento. Defina as configurações do ventilador com base no peso do animal, como descrito anteriormente 11. Volume corrente (ml) = 6,2 x (peso animal em quilogramas) 1.01 e freqüência respiratória = 53,5 x (peso animal em quilogramas) -0.26. </li></ol> 3. A colocação de cateter condutância na câmara LV <ol><li> Abordagem carótida <ol><li> Para garantir a esterilidade, dois conjuntos de instrumentos cirúrgicos estéreis são um utilizado- para incisão cutânea inicial e um para operar no toracotomia. Os instrumentos devem ser descontaminados utilizando um esterilizador seca entre os animais durante uma sessão de um indivíduo cirúrgicoND autoclavados no final de cada dia do procedimento. </li><li> Depois que a pele foi descontaminado com três ciclos de uma clorexidina + álcool esfoliação da pele (solução de álcool / 70% de clorexidina a 0,5%), fazer uma incisão através da carótida direita da mandíbula ao esterno. Dissecar tecido circundante para expor a carótida direita e cortar o nervo vago, que corre ao lado da carótida. </li><li> Coloque uma estéril sutura de seda 6-0 em torno da extremidade distal (afastado do peito) da artéria carótida, gravata e seguro. Coloque dois pontos adicionais sob o proximal da artéria carótida (mais perto do peito) para o primeiro de sutura, vagamente amarrar a sutura meio. Com cuidado, puxe o fio de sutura proximal e garantir apertando-o para a pele. Certifique-se que a artéria carótida foi preso tanto proximal e distal antes de prosseguir. </li><li> Fazer uma pequena incisão na artéria carótida R., proximal em relação ao primeiro fio de sutura, e estendem-se longitudinalmente para o peito. </li><li> Insira a ponta do cateter condutância,previamente embebidas em solução salina quente durante 30 min, para dentro do recipiente através da incisão e fixar o cateter utilizando o meio de sutura. </li><li> Gentilmente avançar o cateter no ventrículo esquerdo através da carótida, enquanto assistia a pressão-volume circuito de rastreamento para assegurar a colocação correta. Nota: a colocação óptima do cateter deve produzir laçadas de pressão e volume que aparecem rectangular (Ver Figura 2). Se alguma resistência à passagem do cateter é encontrado, puxe de volta e avançar novamente com uma leve pressão. Uma rotação suave do cateter pode ajudar no posicionamento para o ventrículo esquerdo. Forçar o cateter condutância pode levar a complicações cardiovasculares ou danos graves para o cateter. </li><li> Ficha de linha de base de pressão-volume circula ~ 10 minutos após a colocação do cateter e alcançar um estado estável (Figura 2). </li></ol></li><li> Abordagem apical <ol><li> Em um rato anestesiado e ventilado, fazer uma incisão from o processo xifóide e corte através da parede torácica lateralmente até que o diafragma é visível. </li><li> Cortar embora o diafragma e visualizar o vértice do coração. </li><li> Inserção do cateter de condutância para o vértice do ventrículo esquerdo através de uma ferida facada agulha (utilizando uma agulha de 25-30 G), até que o eléctrodo proximal é apenas dentro do ventrículo. </li><li> Ficha de linha de base de pressão-volume loops de ~ 10 min após a colocação do cateter e alcançar um estado de equilíbrio. </li></ol></li></ol> 4. Varying pós-carga Usando Transient aórtica Oclusão <ol><li> Para realizar a oclusão da aorta transitória, fazer uma pequena incisão horizontal na parte superior do tórax e dissecar tecido circundante para expor a aorta transversa. </li><li> Coloque uma ligadura de seda 6-0 sob a aorta transversal. Depois de loops de pressão-volume voltaram à linha de base, apertar ambas as extremidades da sutura com a fixação da agulha, suave e lentamente levantar a sutura mais de 1-2 segundos e solte lentamente a tensão. </li&gt; <li> Repetir este procedimento até três gravações separadas óptimas são feitas a partir do mesmo animal. Nota: gravações Optimal deve ter pelo menos 5 ciclos de loop de volume pressão e um aumento constante das pressões sistólica finais durante a aplicação da tensão na sutura (Figura 3A e 3C). </li></ol> 5. Variando Preload Usando Transient Veia Cava Inferior Oclusão (IVC) <ol><li> Para realizar a oclusão da veia cava inferior transitória, fazer uma incisão horizontal abaixo do processo xifóide, sob o diafragma para expor o IVC. </li><li> Coloque uma ligadura de seda 6-0 debaixo do IVC. Depois de loops de pressão-volume voltaram à linha de base, apertar ambas as extremidades da sutura com a fixação da agulha; devagar e com cuidado levantar a sutura sobre 1-2 segundos e solte lentamente a tensão. Oclusão VCI também pode ser realizada pela aplicação de pressão suave usando um cotonete de algodão com ponta. </li><li> Repita este procedimento até três gravações separadas são ideais made do mesmo animal. Nota: gravações Optimal deve ter pelo menos 5 ciclos de loop de volume pressão e uma diminuição constante do diastólica final do ventrículo esquerdo durante a aplicação da tensão na sutura (Figura 4A e 4B). </li></ol> 6. Calibração Saline <ol><li> Na conclusão do estudo, uma (V P) valor de condutância paralela pode ser obter através da injecção de um bolus de 10 ul de solução salina hipertónica (15%) no animal, através da veia jugular (Figura 5A e 5B). Nota: O bólus vai causar um aumento no volume aparente, sem qualquer alteração na pressão. Esta alteração resulta em volume é o resultado de uma alteração na condutância arterial piscina e não devido a um aumento no volume real. Uma diminuição transitória na dP / dt max pode ser observada, como uma solução salina hipertónica tem um efeito inotrópico negativo 13. O calculada V p pode ser inserido em volume de pressão software de análise de ciclo ao longocom os parâmetros de calibração para cuvete e converter de RVUs em microlitros. </li></ol> 7. Calibração Cuvette <ol><li> Para realizar uma calibração cuvete, coloque uma cuvete com poços de diâmetros conhecidos fornecidos pelo fabricante numa almofada de aquecimento ou de um banho de água aquecido a 37 ° C. Preencha os primeiros 4-5 furos com sangue heparinizado fresco quente de ratos submetidos a avaliação hemodinâmica. Nota: A calibração tina permite a avaliação precisa da piscina de sangue do ventrículo esquerdo usando o sangue do mouse e permite a conversão de dados de volume de RVUs para microlitros. </li><li> Inserção do cateter de condutância para o primeiro poço, até que todos os eléctrodos estão submersos. Suavemente mover o cateter dentro do poço, o que irá gerar RVUs variando. </li><li> Grave as alterações de condutância do canal de volume, em RVUs. Seleccionar o maior RVU para a calibração. Nota: O volume dos poços pode ser calculado por 1) usando a equação para o volume de um cilindro, em que o raio é que o poço de cuvete e o comprimento é baseada no comprimento entre os dois eléctrodos de detecção internas ou 2) verificar nas instruções do fabricante. A condutância de saída pode ser correlacionada com os volumes conhecidos para desenvolver uma equação de calibração que converte os dados de RVUs em 11 microlitros. </li></ol> 8. A eutanásia <ol><li> Na conclusão do protocolo, os ratos são sacrificados usando deslocamento cervical sob anestesia. </li><li> A fim de garantir a morte, os ratinhos que têm sido submetidos a deslocação cervical deve ser submetido a um método secundário de eutanásia. Nós usamos sangria sob anestesia, com a colheita de tecido cardíaco para a experimentação, ou toracotomia bilateral sob anestesia. </li></ol> 9. Análise de Dados usando Volume Pressão laço Analysis Software <ol><li> Calcule V p de Calibração Saline <ol><li> Selecionar pressão e volume laços obtido durante a ejecção de uma solução salina hipertónica (Figura 5A e 5B) </li><li> Exportação dos ciclos para o volume de pressão de software de análise. Escolha a opção para a calibração Saline (Figura 5C) </li><li> Grave o valor V p calculado (Figura 5D). Nota: O valor de p V é calculado por meio da identificação do cruzamento do volume 1) Fim-diastólica vs. volume sistólico final da calibração salina e 2) linha de volume volume = End-End-sistólica diastólica. O cruzamento destas linhas fornece a V P, o qual é calculado o volume por pressão do software de análise de ciclo. </li></ol></li><li> Digite condutância ao volume (RVU) em relação opções de canais de volume (Figura 1F) </li><li> Linha de Base relação ciclo pressão-volume Meça <ol><li> Selecione 8-10 ciclos cardíacos de canais de pressão e volume uma vez atingido o estado estacionário (Figura 2A) ea exportação para a análise suaveware. Identificar 5-6 laços expiratória final (Figura 2B) </li><li> Use o valor p V para corrigir a condutância paralela. Escolha a opção &quot;estado estacionário&quot; e gerar uma tabela de resumo hemodinâmico (Figura 2C) </li></ol></li><li> Medida Relação ciclo pressão-volume durante a constrição aórtica <ol><li> Selecione 8-10 ciclos cardíacos de canais de pressão e volume que correspondem a constrição aórtica antes do aumento de diastólica final (Figura 3A) e exportação de software de análise. Identificar 5-6 laços expiratória final (Figura 3C) </li><li> Selecione análise &quot;Contractilidade&quot; (Figura 3B), que irá calcular o volume Pressão Relação sistólico final (ESPVR) </li></ol></li><li> Medida Relação ciclo pressão-volume durante IVC constrição <ol><li> Selecione 8-10 ciclos cardíacos de canais de pressão e volume que correspondem a IVC constrição (Figure 4A) e exportação de software de análise. Identificar 5-6 laços expiratória final (Figura 4B) </li><li> Selecione análise &quot;Contractilidade&quot; (Figura 3B), que irá calcular Preload recrutável Trabalho Curso (PRSW) (Figura 4C), máxima dP / dt vs EDV (Figura 4D), bem como a ESPVR e relação do volume a pressão diastólica final (Figura 4E) </li></ol></li></ol>

<ol>
	<li>Clark, J. E., Marber, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+in+pressure-volume+catheter+technology+-+stress+remodelling+after+infarction.">Advancements in pressure-volume catheter technology - stress remodelling after infarction.</a> Exp Physiol. 98 (3), 614-621 (2013).</li><li>Sunagawa, K., Maughan, W. L., Burkhoff, D., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+interaction+with+arterial+load+studied+in+isolated+canine+ventricle.">Left ventricular interaction with arterial load studied in isolated canine ventricle.</a> Am J Physiol. 245 (Pt 1), H773-H780 (1983).</li><li>Suga, H., Sagawa, K., Demer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+instantaneous+pressure+in+canine+left+ventricle.+Time+and+volume+specification.">Determinants of instantaneous pressure in canine left ventricle. Time and volume specification.</a> Circ Res. 46 (2), 256-263 (1980).</li><li>Suga, H., Sagawa, K., Shoukas, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Load+independence+of+the+instantaneous+pressure-volume+ratio+of+the+canine+left+ventricle+and+effects+of+epinephrine+and+heart+rate+on+the+ratio.">Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio.</a> Circ Res. 32 (3), 314-322 (1973).</li><li>Kass, D. 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=Improved+left+ventricular+mechanics+from+acute+VDD+pacing+in+patients+with+dilated+cardiomyopathy+and+ventricular+conduction+delay.">Improved left ventricular mechanics from acute VDD pacing in patients with dilated cardiomyopathy and ventricular conduction delay.</a> Circulation. 99 (12), 1567-1573 (1999).</li><li>Kass, D. 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=Diastolic+Compliance+of+Hypertrophied+Ventricle+Is+Not+Acutely+Altered+by+Pharmacological+Agents+Influencing+Active+Processes.">Diastolic Compliance of Hypertrophied Ventricle Is Not Acutely Altered by Pharmacological Agents Influencing Active Processes.</a> Annals of Internal Medicine. 119 (6), 466-473 (1993).</li><li>Georgakopoulos, D., 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=In+vivo+murine+left+ventricular+pressure-volume+relations+by+miniaturized+conductance+micromanometry.">In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry.</a> Am J Physiol. 274 (4 pt 2), H1416-H1422 (1998).</li><li>Cingolani, O. H., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-volume+relation+analysis+of+mouse+ventricular+function.">Pressure-volume relation analysis of mouse ventricular function.</a> Am J Physiol Heart Circ Physiol. 301 (6), H2198-H2206 (2011).</li><li>Baan, 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=Continuous+Measurement+of+Left-Ventricular+Volume+in+Animals+and+Humans+by+Conductance+Catheter.">Continuous Measurement of Left-Ventricular Volume in Animals and Humans by Conductance Catheter.</a> Circulation. 70 (5), 812-823 (1984).</li><li>Pearce, J. A., Porterfield, J. E., Larson, E. R., Valvano, J. W., Feldman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+considerations+in+catheter+based+estimation+of+left+ventricular+volume.">Accuracy considerations in catheter based estimation of left ventricular volume.</a> Conf Proc IEEE Eng Med Biol Soc. 2010, 3556-3558 (2010).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nature Protocols. 3 (9), 1422-1434 (1038).</li><li>Hanusch, C., Hoeger, S., Beck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+of+small+rodents+during+magnetic+resonance+imaging.">Anaesthesia of small rodents during magnetic resonance imaging.</a> Methods. 43 (1), 68-78 (2007).</li><li>Georgakopoulos, D., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+parallel+conductance+by+dual-frequency+conductance+catheter+in+mice.">Estimation of parallel conductance by dual-frequency conductance catheter in mice.</a> Am J Physiol Heart Circ Physiol. 279 (1), H443-H450 (2000).</li><li>Esposito, G., 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=Increased+myocardial+contractility+and+enhanced+exercise+function+in+transgenic+mice+overexpressing+either+adenylyl+cyclase+5+or+8.">Increased myocardial contractility and enhanced exercise function in transgenic mice overexpressing either adenylyl cyclase 5 or 8.</a> Basic Res Cardiol. 103 (1), 22-30 (2008).</li><li>Kohout, T. 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=Augmentation+of+cardiac+contractility+mediated+by+the+human+beta(3)-adrenergic+receptor+overexpressed+in+the+hearts+of+transgenic+mice.">Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.</a> Circulation. 104 (20), 2485-2491 (2001).</li><li>Kim, K. S., 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=beta-Arrestin-biased+AT1R+stimulation+promotes+cell+survival+during+acute+cardiac+injury.">beta-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury.</a> Am J Physiol Heart Circ Physiol. 303 (8), H1001-H1010 (2012).</li><li>Suga, H., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+Interrelationship+between+Instantaneous+Ventricular+Pressure-Volume+Ratio+and+Myocardial+Force-Velocity+Relation.">Mathematical Interrelationship between Instantaneous Ventricular Pressure-Volume Ratio and Myocardial Force-Velocity Relation.</a> Annals of Biomedical Engineering. 1 (2), 160-181 (1972).</li><li>Suga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+energetics.">Ventricular energetics.</a> Physiol Rev. 70 (2), 247-277 (1990).</li><li>Kass, D. 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=Influence+of+contractile+state+on+curvilinearity+of+in+situ+end-systolic+pressure-volume+relations.">Influence of contractile state on curvilinearity of in situ end-systolic pressure-volume relations.</a> Circulation. 79 (1), 167-178 (1989).</li><li>Little, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+left+ventricular+dP/dtmax-end-diastolic+volume+relation+in+closed-chest+dogs.">The left ventricular dP/dtmax-end-diastolic volume relation in closed-chest dogs.</a> Circ Res. 56 (6), 808-815 (1985).</li><li>Glower, D. D., 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=Linearity+of+the+Frank-Starling+relationship+in+the+intact+heart:+the+concept+of+preload+recruitable+stroke+work.">Linearity of the Frank-Starling relationship in the intact heart: the concept of preload recruitable stroke work.</a> Circulation. 71 (5), 994-1009 (1985).</li><li>Sharir, T., 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=Ventricular+systolic+assessment+in+patients+with+dilated+cardiomyopathy+by+preload-adjusted+maximal+power.+Validation+and+noninvasive+application.">Ventricular systolic assessment in patients with dilated cardiomyopathy by preload-adjusted maximal power. Validation and noninvasive application.</a> Circulation. 89 (5), 2045-2053 (1994).</li><li>Baan, J., Van der Velde, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+left+ventricular+end-systolic+pressure-volume+relation+to+type+of+loading+intervention+in+dogs.">Sensitivity of left ventricular end-systolic pressure-volume relation to type of loading intervention in dogs.</a> Circ Res. 62 (6), 1247-1258 (1988).</li><li>Wei, C. L., 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=Volume+catheter+parallel+conductance+varies+between+end-systole+and+end-diastole.">Volume catheter parallel conductance varies between end-systole and end-diastole.</a> IEEE Trans Biomed Eng. 54 (8), 1480-1489 (2007).</li><li>Porterfield, J. E., 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=Dynamic+correction+for+parallel+conductance,+GP,+and+gain+factor,+alpha,+in+invasive+murine+left+ventricular+volume+measurements.">Dynamic correction for parallel conductance, GP, and gain factor, alpha, in invasive murine left ventricular volume measurements.</a> J Appl Physiol (1985). 107 (6), 1693-1703 (2009).</li></ol>

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Nós descrevemos um método para perfoming análise do lacete pressão-volume usando um cateter de condutância em camundongos, para derivar análises abrangentes, tanto a contratilidade cardíaca e relaxamento. Suga, Sagawa e seus colegas utilizaram pressão-volume loops para definir medidas de contratilidade cardíaca, especificamente a inclinação da ESPVR, ou a elastância sistólica final (es E), e E max. Elastância, definida pela relação de pressão para o volume (P / V), varia ao longo d...

Análise de ciclo de volume pressão cardíaca fornece informações detalhadas da função cardíaca e são o padrão ouro para avaliação funcional 1. Embora as técnicas de imagem, como a ecocardiografia ou a ressonância magnética cardíaca fornecer medidas funcionais, estas medidas são altamente dependentes de condições de carga. Medidas independentes de Carga, de contratilidade cardíaca e relaxamento requerem medições dinâmicas da pressão ventricular e relação do volume em um intervalo de pré-carga e pós-carga. Esta compreensão da relação pressão-volume decorre do trabalho inovador de Sagawa e colegas 2,3. Eles demonstraram em ex vivo corações caninos perfundidos que o loop de pressão-volume derivados medidas de contratilidade foram independentes de condições de carga 4. Na aplicação in vivo destas análises tornou-se possível, com o desenvolvimento de cateteres de condutância na década de 1980. Este avanço técnico permitiu Kass e colegas para realizar a análise de ciclo de pressão-volume em humanos 5,6. Miniaturização dos cateteres de condutância e melhorias nas técnicas cirúrgicas no final de 1990 de 7 feita a análise da função cardíaca roedor viável, permitindo estudos genéticos e farmacológicos para ser realizado. Este avanço, desde então, levar ao uso generalizado de análise de ciclo de pressão-volume e tem gerado uma grande quantidade de conhecimento sobre fisiologia cardíaca mamíferos. Um conceito fundamental na utilização de cateteres de condutância e a interpretação dos dados obtidos a partir dele, é a relação entre o volume e a condutância. A condutância é inversamente relacionada com a tensão, que é medida através de um cateter com eléctrodos colocados proximal, geralmente colocada por baixo da válvula aórtica, e distalmente, no apex LV 8. Alterações na tensão ou condutância são medidos por mudanças na corrente que flui de proximal para distal do eletrodo. Embora a piscina de sangue contribuems significativamente a condutância, a contribuição da parede ventricular, denominado condutância paralela (V P), a condutância medido deve ser subtraído para se obter medições do volume do VE absolutos. Os métodos para efectuar esta correcção, chamado de calibração solução salina, são discutidos no protocolo abaixo. A relação matemática entre a condutância e de volume, descrito por Baan e colegas, é que o volume = 1 / α; (ρ L 2) (GG p), onde fator de correção α = campo uniforme, ρ = resistividade sangue, L = distância entre os eletrodos, G = condutância e G p = condutância não-sangue 9. De nota, o fator de correção de campo uniforme em camundongos aproxima de 1,0 devido a pequenos volumes de câmara 10. Juntamente com transdutores de pressão, o cateter condutância fornece dados de pressão e volume simultânea em tempo real. Pressu cardíacare-análise do volume apresenta determinadas vantagens sobre outras medidas da função cardíaca, já que permitem a medição da função ventricular independente das condições de carga e de frequência cardíaca. Índices cardíacos independente de carregamento específicos de contratilidade incluem: sistólico final do volume de relação de pressão (ESPVR), d P / d t max relação volume de -end-diastólica, elastância máxima (E máx) e pré-carga de trabalho do curso recrutável (PRSW). Uma medida independente de carga da função diastólica é a relação do volume a pressão diastólica final (EDPVR) 11. O protocolo a seguir descreve o comportamento de análise de ciclo de volume pressão cardíaca, usando tanto um carótida e uma abordagem apical. Embora a metodologia para executar estes estudos foram descritas em pormenor anteriormente 8,11, iremos analisar os passos chave para obter medições de pressão-volume preciso, incluindo tanto a correcção de solução salina e de calibração cuvete, e fornecer uma demonstração visual de Tsprocedimentos e. Pesquisas com animais realizados para este estudo foi tratado de acordo com os protocolos aprovados e os regulamentos de protecção dos animais do Comité de Conservação e Uso do Duke University Medical Center animal Institucional.

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			<td>1.4F conductance catheter&#160;</td>
			<td>ADInstruments Inc</td>
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			<td>427401</td>
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			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
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			<td>427411</td>
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			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
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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.

Análise de ciclo de pressão-volume pode ser usado para medir a função cardíaca em camundongos geneticamente modificados 14,15 ou camundongos submetidos a estudos de drogas 16. Lacetes de volume pressão representativos são fornecidos a partir de trabalhos publicados anteriormente 16 investigar o efeito de beta-arrestina inclinado AT1R ligando, TRV120023. Para testar se TRV120023 afeta a função cardíaca in vivo, análise do lacete pressão-volume foi realizada em camundo...

Watch this Scientific Journal Video about Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice at JoVE.com

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters 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 ...

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

19.3K visualizações.  Duke University Medical Center. 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. 

Vídeo: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Biologia

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioengenharia Humanos Networks microvascular em camundongos imunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expressão e purificação do Fibrose Cística proteína transmembranar regulador da condutância na Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Medição de condutividade hidráulica da folha e da condutância estomática e as respostas a irradiância e Desidratação Usando a evaporação Método Flux (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital da Microcirculação no músculo Rato Cremaster de Análise de Migração Stem Peripheral celular

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purificação da fibrose cística condutância transmembrana Regulador proteína expressa em<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Uma nova técnica de Análise Quantitativa de perda de cabelo em ratos usando Análise Tons de Cinza

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Relacionando condutância estomática a Folha atributos funcionais

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Medidas de contratilidade no isolado músculos papilares de Investigação de Cardiac inotropia em Ratos

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Medição do volume Pressão Loops no mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...