Ce travail est soutenu par l&#39;American Heart Association 14FTF20370058 (DMA) et le NIH T32 HL007101-35 (DMA).

1. Préparation de la conductance cathéter et étalonnage de pression <ol><li> Connecter conductance cathéter au module de cathéter hémodynamique. Calibrer électroniquement mesures de pression et de volume en enregistrant pression préréglée et le volume réglé sur le module de cathéter. Enregistrer un tracé de 0 mm Hg et 25 mm Hg (figure 1A) et attribuer des tensions aux deux tracés de pression (figure 1B et 1C). De même, enregistrer un tracé de volume de 5 RVU et 25 RVU (figure 1D) et attribuer des tensions aux deux tracés de volume (de 1e et 1f). </li><li> Confirmez l&#39;étalonnage de pression électronique avec un calibrage manuel de pression, à l&#39;aide d&#39;un sphygmomanomètre à colonne de mercure. Monter le port tensiomètre brassard avec un robinet 3 voies. Remplir un système de valve hémostatique 3-port, souvent utilisé pour l&#39;angioplastie coronarienne, avec de l&#39;eau en utilisant la RT orifice latéral. <ol><li> Placez l&#39;extrémité d&#39;un cathéter de conductance dans la valve hémostatique rempli de fluide et fixer doucement l&#39;esprithout vriller cathéter. Branchez la valve hémostatique à l&#39;tensiomètre et gonfler à 200 mmHg et verrouiller robinet à 3 voies. Examiner si la pression mesurée correspond à la pression gonflé sur le tensiomètre. </li></ol></li><li> Lieu cathéter dans une solution saline chauffée à 37 ° C qui est au niveau de la pression de champ opératoire et à mesurer. Réglez le contrôle de la pression jusqu&#39;à ce que les pressions enregistrées est à zéro. </li></ol> 2. Anesthésie / intubation <ol><li> Administrer la kétamine / xylazine (80-100 / 10 mg kg -1) comme une injection intrapéritonéale. Remarque: Les agents anesthésiques alternatifs peuvent être utilisés. Une liste complète des anesthésiques sont fournis dans les commentaires précédents de cette technique 11,12. Anesthésie adéquate peut être confirmée par douce pincement de la queue. </li><li> Une fois anesthésié, de se raser le cou et la poitrine avec les tondeuses et place sur un coussin chauffant. Maintenir la température rectale de la souris à 36,5 à 37,5 ° C. La température du corps Lowse traduira par des taux cardiaques déprimés. Appliquer une pommade pour les yeux pour prévenir la sécheresse </li><li> Faire une incision médiane dans le cou et de disséquer les muscles de la trachée pour exposer la trachée. Placer une sonde endotrachéale par la bouche, tout en visualisant la trachée pour assurer l&#39;intubation, et se connecter à l&#39;appareil respiratoire. </li><li> Maintenir la souris sur le ventilateur pendant la procédure. Les réglages du ventilateur réglée sur la base du poids de l&#39;animal comme décrit précédemment 11. Volume courant (ml) = 6,2 x (poids de l&#39;animal en kilogrammes) et la fréquence respiratoire 1.01 = 53,5 x (poids de l&#39;animal en kilogrammes) -0.26. </li></ol> 3. Mise en place de la conductance cathéter dans la chambre LV <ol><li> Approche de la carotide <ol><li> Pour assurer la stérilité, deux ensembles d&#39;instruments chirurgicaux stériles sont utilisé- un pour incision cutanée initiale et un à opérer dans la thoracotomie. Les instruments doivent être décontaminés à l&#39;aide d&#39;un stérilisateur à sec entre les animaux au cours d&#39;une session individuelle chirurgicale unee autoclave à la fin de chaque jour chirurgicale. </li><li> Après la peau a été décontaminé avec trois cycles d&#39;une chlorhexidine + alcool gommage de la peau (solution d&#39;alcool / 70% de chlorhexidine 0,5%), faire une incision sur la carotide droite de la mandibule au sternum. Disséquer les tissus environnants pour exposer la carotide droite, et couper le nerf vague qui longe la carotide. </li><li> Placez une suture de soie 6-0 stérile autour de l&#39;extrémité distale (loin de la poitrine) de l&#39;artère carotide, une cravate et sécurisé. Placer deux sutures supplémentaires sous l&#39;extrémité proximale de l&#39;artère carotide (plus près de la poitrine) pour le premier fil de suture, la suture attachant lâchement milieu. Tirez doucement sur la suture proximale et sécuriser en le serrant à la peau. Assurez-vous que l&#39;artère carotide a été serré à la fois proximale et distale avant de poursuivre. </li><li> Faire une petite incision dans l&#39;artère carotide R., proximale à la première suture, et prolonger longitudinalement vers la poitrine. </li><li> Insérer la pointe conductance de cathéter,préalablement trempées dans l&#39;eau chaude salée pendant 30 min, dans le récipient par l&#39;incision et fixer le cathéter en utilisant la suture milieu. </li><li> Avancer doucement le cathéter dans le ventricule gauche à travers la carotide, tout en regardant la boucle pression-volume traçage pour assurer le placement correct. Note: L&#39;installation optimale du cathéter devrait donner boucles pression-volume qui apparaissent rectangulaire (voir figure 2). Si aucune résistance au passage du cathéter est rencontrée, tirez doucement en arrière et avancer de nouveau avec une légère pression. Une légère rotation du cathéter peut aider avec le placement dans le ventricule gauche. Forcer le cathéter de conductance peut entraîner des complications ou des dommages au cathéter cardiovasculaires graves. </li><li> Base de registre pression-volume boucles ~ 10 min après la pose de cathéter et la réalisation d&#39;un état ​​d&#39;équilibre (Figure 2). </li></ol></li><li> Approche apicale <ol><li> Dans une souris anesthésiée et ventilé, faire une incision from le processus de xiphoïde et couper à travers la paroi thoracique latéralement jusqu&#39;à ce que la membrane est visible. </li><li> Couper si le diaphragme et visualiser l&#39;apex du cœur. </li><li> Insérer le cathéter de conductance dans l&#39;apex du ventricule gauche à travers une aiguille blessure par arme blanche (en utilisant une aiguille de 25 à 30 G), jusqu&#39;à ce que l&#39;électrode proximale est juste à l&#39;intérieur du ventricule. </li><li> Base de registre pression-volume boucles ~ 10 min après la pose de cathéter et la réalisation d&#39;un état d&#39;équilibre. </li></ol></li></ol> 4. variant Postcharge Utilisation transitoire Occlusion aortique <ol><li> Pour effectuer une occlusion aortique transitoire, faire une petite incision horizontale dans la partie supérieure du thorax et de disséquer les tissus environnants pour exposer l&#39;aorte transversale. </li><li> Placer une ligature de soie 6-0 sous l&#39;aorte transverse. Après boucles pression-volume sont retournés à la ligne de base, serrer les deux extrémités de la suture avec une pince d&#39;aiguille, doucement et lentement augmenter la suture sur 1-2 secondes, puis relâchez lentement la tension. </li&gt; <li> Répétez cette procédure jusqu&#39;à trois enregistrements distincts optimales sont fabriqués à partir du même animal. Remarque: Optimal enregistrements doivent avoir au moins 5 volumes de pression cycles de boucle et une augmentation constante des pressions systoliques finaux au cours de l&#39;application d&#39;une tension sur le fil de suture (figure 3A et 3C). </li></ol> 5. variant précharge Utilisation transitoire veine cave inférieure Occlusion (IVC) <ol><li> Pour effectuer transitoire veine cave inférieure occlusion, faire une incision horizontale en dessous de la pointe du sternum, sous le diaphragme pour exposer la VCI. </li><li> Placez une ligature 6-0 de soie sous le IVC. Après boucles pression-volume sont retournés à la ligne de base, serrer les deux extrémités de la suture avec une pince d&#39;aiguille; doucement et lentement augmenter la suture sur 1-2 secondes, puis relâchez lentement la tension. IVC occlusion peut également être effectuée par l&#39;application en douceur de la pression aide d&#39;un coton-tige pointe. </li><li> Répétez cette procédure jusqu&#39;à trois enregistrements distincts sont optimales made du même animal. Remarque: Optimal enregistrements doivent avoir au moins 5 volumes de pression cycles de boucle et une baisse constante des pressions ventriculaires gauche de fin de diastole lors de l&#39;application de la tension sur le fil de suture (figure 4A et 4B). </li></ol> 6. Saline Calibration <ol><li> A la fin de l&#39;étude, une conductance parallèle (V p) valeur peut être obtenir par injection d&#39;un bolus de 10 pl d&#39;une solution saline hypertonique (15%) dans l&#39;animal à travers la veine jugulaire (figure 5A et 5B). Remarque: Ce bolus provoque une augmentation du volume apparent, sans modification de la pression. Ce changement apparent en volume est le résultat d&#39;un changement dans le sang piscine conductance plutôt que due à une augmentation réelle du volume. Une baisse transitoire de la dP / dt max peut être observé, comme une solution saline hypertonique a un effet inotrope négatif 13. Le V p calculé peut être entré dans le volume de pression logiciel d&#39;analyse de boucle le longavec les paramètres d&#39;étalonnage de la cuvette et à convertir les UVR en microlitres. </li></ol> 7. Cuvette Calibration <ol><li> Pour effectuer un étalonnage de la cuvette, placer une cuvette avec des puits de diamètres connus fournis par le fabricant sur un coussin chauffant ou un bain d&#39;eau chauffée à 37 ºC. Remplissez les 4-5 premiers trous avec sang héparine chaud frais de souris l&#39;objet d&#39;évaluations hémodynamiques. Remarque: une cuvette de calibrage permet une évaluation précise de pool sanguin du ventricule gauche en utilisant le sang de souris et permet la conversion de données de volume de UVR à microlitres. </li><li> Insérer le cathéter de la conductance dans le premier puits, jusqu&#39;à ce que toutes les électrodes sont immergées. Déplacez doucement le cathéter dans le puits, ce qui va générer RVUs variable. </li><li> Enregistrez les modifications de conductance dans le volume du canal dans RVUs. Sélectionnez la plus haute RVU pour l&#39;étalonnage. Remarque: Le volume des puits peut être calculée soit par 1) en utilisant l&#39;équation pour le volume d&#39;un cylindre, où le rayon est celui du puits de cuve et la longueur est basée sur la longueur entre les deux électrodes de détection interne ou 2) la vérification dans les instructions du fabricant. La sortie de la conductance peut être corrélée avec les volumes connus pour développer une équation d&#39;étalonnage qui convertit les données à partir de 11 microlitres dans UVR. </li></ol> 8. euthanasie <ol><li> À la conclusion du protocole, les souris sont euthanasiées par dislocation cervicale sous anesthésie. </li><li> Afin d&#39;assurer la mort, les souris ayant subi une dislocation cervicale doivent subir une méthode secondaire de l&#39;euthanasie. Nous utilisons exsanguination sous anesthésie, avec la récolte du tissu cardiaque pour l&#39;expérimentation, ou thoracotomie bilatérale sous anesthésie. </li></ol> 9. Analyse des données en utilisant un volume de pression boucle logiciel d&#39;analyse <ol><li> Calculer V p de calibrage Saline <ol><li> Sélectionnez pression et volumoi boucles obtenu lors de l&#39;éjection de solution saline hypertonique (figure 5A et 5B) </li><li> Exporter boucles de logiciel d&#39;analyse de volume de pression. Choisissez l&#39;option pour l&#39;étalonnage de Saline (Figure 5C) </li><li> Enregistrez la valeur V de p calculée (Figure 5D). Remarque: La valeur de p de V est calculée en identifiant l&#39;intersection du volume 1) Fin-diastolique vs le volume en fin de systole de l&#39;étalonnage de la solution saline et 2) la ligne de volume volume = Fin-Fin systolique-diastolique. L&#39;intersection de ces lignes offre la V p, qui est calculée par le logiciel d&#39;analyse de boucle de volume de pression. </li></ol></li><li> Entrez la conductance au volume (ERF) relation dans les options de canal de volume (figure 1F) </li><li> Mesurer Baseline pression-volume relation de boucle <ol><li> Sélectionnez 8-10 cycles cardiaques provenant des canaux de pression et de volume une fois l&#39;état d&#39;équilibre a été atteint (figure 2A) et l&#39;exportation à l&#39;analyse douceWare. Identifier 5-6 boucles fin d&#39;expiration (figure 2B) </li><li> Utilisez la valeur de p de V pour corriger la conductance parallèle. Choisissez l&#39;option &quot;Steady State&quot; et de générer un tableau récapitulatif hémodynamique (figure 2C) </li></ol></li><li> Mesure pression-volume Relation de boucle lors de constriction aortique <ol><li> Sélectionnez 8-10 cycles cardiaques provenant des canaux de pression et de volume qui correspondent à une constriction aortique avant l&#39;augmentation des pressions de fin de diastole (figure 3A) et l&#39;exportation vers des logiciels d&#39;analyse. Identifier 5-6 boucles fin d&#39;expiration (figure 3C) </li><li> Sélectionnez l&#39;analyse &quot;contractilité&quot; (figure 3B), qui calcule le volume de pression Relation en fin de systole (ESPVR) </li></ol></li><li> Mesure pression-volume Relation de boucle pendant IVC constriction <ol><li> Sélectionnez 8-10 cycles cardiaques provenant des canaux de pression et de volume qui correspondent à IVC constriction (Figure 4A) et l&#39;exportation de logiciels d&#39;analyse. Identifier 5-6 boucles fin d&#39;expiration (figure 4B) </li><li> Sélectionnez l&#39;analyse &quot;contractilité&quot; (figure 3B), qui calculera travail systolique recrutable précharge (PRSW) (figure 4C), dp / dt maximale vs EDV (figure 4D), ainsi que la ESPVR et la relation pression-volume télédiastolique (Figure 4E) </li></ol></li></ol>

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Les auteurs ont rien à révéler.

Nous décrivons une méthode pour perfoming analyse de la boucle pression-volume en utilisant un cathéter de conductance chez la souris, de tirer des analyses complètes des deux contractilité cardiaque et de détente. Suga, Sagawa et collègues utilisées pression-volume boucles de définir les mesures de contractilité cardiaque, en particulier la pente de la ESPVR, ou l&#39;élastance télésystolique (E s), et E max. Élastance, défini par le rapport de la pression de volume (P / V), varie s...

Cardiac analyse de la boucle pression-volume fournit des informations détaillées de la fonction cardiaque et sont l&#39;étalon-or pour l&#39;évaluation fonctionnelle 1. Bien que les techniques d&#39;imagerie telles que l&#39;échographie ou l&#39;IRM cardiaque fournissent des mesures fonctionnelles, ces mesures sont très dépendants des conditions de chargement. Mesures d&#39;indépendant de la charge de la contractilité cardiaque et la relaxation exigent des mesures dynamiques de la pression ventriculaire et la relation de volume sur une plage de précharge et la postcharge. Cette compréhension de la relation pression-volume provient du travail de pionnier de Sagawa et ses collègues 2,3. Ils ont démontré ex vivo coeurs canins perfusés que les dérivés mesures de contractilité de boucle pression-volume étaient indépendants des conditions de chargement 4. En application de ces analyses in vivo n&#39;a été rendu possible par le développement de cathéters de conductance dans les années 1980. Ce progrès technique a permis Kass et collègues pour effectuer pression-volume analyse en boucle chez les humains 5,6. La miniaturisation des cathéters et des améliorations dans les techniques chirurgicales conductance à la fin des années 1990, a fait sept analyse de la fonction cardiaque de rongeur possible, ce qui permet pour les études génétiques et pharmacologiques à effectuer. Cette avance a depuis conduit à l&#39;utilisation généralisée de l&#39;analyse de la boucle pression-volume et a suscité beaucoup de perspicacité dans la physiologie cardiaque des mammifères. Un concept clé dans l&#39;utilisation de cathéters de conductance et l&#39;interprétation des données obtenues à partir de ce est la relation entre le volume et la conductance. La conductance est inversement proportionnelle à la tension, qui est mesurée en utilisant un cathéter avec des électrodes placées de manière proximale, habituellement placés au-dessous de la valve aortique, et distalement, au sommet LV 8. Changements de la tension ou de la conductivité sont mesurés par les variations du courant qui en découlent proximale à l&#39;électrode distale. Bien que la piscine de sang contribuents de manière significative à la conductance, la contribution de la paroi ventriculaire, appelée la conductance parallèle (V p), de la conductance mesurée doit être soustraite pour obtenir des mesures de volume de LV absolus. Les méthodes pour effectuer cette correction, appelé un étalonnage de solution saline, sont discutés dans le protocole ci-dessous. La relation mathématique entre la conductance et le volume, décrit par Baan et ses collègues, est que le volume = 1 / α; (ρ L 2) (GG p), où α = champ uniforme facteur de correction, ρ = résistivité du sang, L = distance entre les électrodes, G = conductance G et p = conductance non-sang neuf. Il faut noter que le champ uniforme facteur de correction en se rapproche de 1,0 souris en raison du faible volume de la chambre 10. Couplé avec des transducteurs de pression, le cathéter à conductance fournit des données de pression et de volume simultanée en temps réel. PRESSU cardiaquere volume analyse présente des avantages particuliers sur les autres mesures de la fonction cardiaque, car ils permettent la mesure de la fonction ventriculaire indépendante des conditions de charge et de la fréquence cardiaque. Indices spécifiques indépendants de la charge cardiaques de la contractilité comprennent: en fin de systole relation pression-volume (ESPVR), d P / d t max relation de volume -end-diastolique, élastance maximale (E max) et de la précharge du travail systolique recrutable (PRSW). Une mesure de la fonction diastolique indépendant de la charge est le rapport de volume de fin de diastole de pression (EDPVR) 11. Le protocole décrit le déroulement de l&#39;analyse cardiaque en boucle pression-volume, en utilisant à la fois une carotide et une approche apicale. Bien que la méthodologie pour effectuer ces études ont été décrites en détail précédemment 8,11, nous passerons en revue les principales étapes pour obtenir des mesures de pression-volume précis, y compris à la fois la correction de solution saline et l&#39;étalonnage de la cuvette, et de fournir une démonstration visuelle de Thesles procédures de e. Recherche sur des animaux effectués pour cette étude a été gérée selon des protocoles approuvés et aux règles de protection des animaux du comité soin et l&#39;utilisation des animaux institutionnel de Duke University Medical Center.

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			<td>AnaSed (xylazine)&#160;</td>
			<td>Lloyd Laboratories</td>
			<td>NADA no. 139-236</td>
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			<td>Ketaset (ketamine)</td>
			<td>Pfizer</td>
			<td>440842</td>
			<td>Anesthetic</td>
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			<td>VIP3000</td>
			<td>Matrx Medical Inc.</td>
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			<td>Anesthesia machine</td>
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			<td>Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>Model 683</td>
			<td>Surgical Equipment</td>
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			<td>Tubing kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1049</td>
			<td>Surgical Equipment</td>
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			<td>Homeothermic Blanket&#160;</td>
			<td>Kaz Inc.</td>
			<td>5628</td>
			<td>Surgical Equipment</td>
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			<td>Stereo microscope</td>
			<td>Carl Zeiss Optical Inc.</td>
			<td>Stemi 2000</td>
			<td>Surgical Equipment</td>
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			<td>Illuminator</td>
			<td>Cole&#8211;Parmer</td>
			<td>41720</td>
			<td>Surgical Equipment</td>
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			<td>Dumont no. 55 Dumostar Forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11295-51</td>
			<td>Surgical Instruments</td>
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			<td>Graefe forceps, curved&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11052-10</td>
			<td>Surgical Instruments</td>
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			<td>Moria MC31 forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11370-31</td>
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			<td>Mayo scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14512-15</td>
			<td>Surgical Instruments</td>
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			<td>Iris scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14041-10</td>
			<td>Surgical Instruments</td>
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			<td>Halsey needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12501-13</td>
			<td>Surgical Instruments</td>
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			<td>Olsen&#8211;Hegar needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12002-12</td>
			<td>Surgical Instruments</td>
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			<td>spring scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>15610-08</td>
			<td>Surgical Instruments</td>
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		<tr>
			<td>disposable underpads</td>
			<td>Kendall/Tyco Healthcare</td>
			<td>1038</td>
			<td>Surgical Supplies</td>
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			<td>Sterile gauze sponges, sterile&#160;</td>
			<td>Dukal</td>
			<td>62208</td>
			<td>Surgical Supplies</td>
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			<td>Cotton-tipped applicators, sterile&#160;</td>
			<td>Solon</td>
			<td>368</td>
			<td>Surgical Supplies</td>
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			<td>Surgical suture,&#160; silk, 6-0&#160;</td>
			<td>DemeTECH</td>
			<td>FT-639-1</td>
			<td>Surgical Supplies</td>
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			<td>1 cc Insulin syringes&#160;</td>
			<td>Becton Dickenson</td>
			<td>329412</td>
			<td>Surgical Supplies</td>
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			<td>Access-9&#8482; Hemostasis Valve</td>
			<td>Merit Medical&#160;</td>
			<td>MAP111</td>
			<td>Hemodynamic equipment</td>
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			<td>Sphygmomanometer</td>
			<td>Baumanometer</td>
			<td>320</td>
			<td>Hemodynamic equipment</td>
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			<td>Millar PV system MPVS-300/400 or MPVS Ultra (includes calibration cuvette)</td>
			<td>ADInstruments Inc</td>
			<td></td>
			<td>Hemodynamic equipment</td>
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			<td>1.4F conductance catheter&#160;</td>
			<td>ADInstruments Inc</td>
			<td>SPR-839</td>
			<td>Hemodynamic equipment</td>
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			<td>PowerLab 4/30 with Chart Pro</td>
			<td>ADInstruments Inc.</td>
			<td>ML866/P</td>
			<td>Hemodynamic software</td>
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			<td>animal clipper</td>
			<td>Wahl</td>
			<td>8787-450A</td>
			<td>Miscellaneous</td>
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			<td>Intradermic tubing PE-10 (Becton Dickinson, cat. no. )</td>
			<td>Becton Dickenson</td>
			<td>427401</td>
			<td>Miscellaneous</td>
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			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
			<td>Becton Dickenson</td>
			<td>427411</td>
			<td>Miscellaneous</td>
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			<td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
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			<td>0.9% (wt/vol) sodium chloride injection, USP , cat. no. )</td>
			<td>Hospira</td>
			<td>NDC no. 0409-4888-50</td>
			<td>Miscellaneous</td>
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			<td>Surgical tape</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
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			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
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			<td>Miscellaneous</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.

Analyse en boucle pression-volume peut être utilisée pour mesurer la fonction cardiaque chez des souris génétiquement modifiées ou des souris subissant 14,15 études sur les médicaments 16. Représentant boucles de volume de pression sont prévus à partir de travaux déjà publiés 16 étudier l&#39;effet de la ß-Arrestin biaisé ligand AT1R, TRV120023. Pour tester si TRV120023 affecte la fonction cardiaque in vivo, une analyse en boucle pression-volume a été effectué...

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 pression-volume Analyse boucle aide de la conductance cathéters chez la souris

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 vues.  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. 

Vidéo: 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.

Patch Clamp et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

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

Recherche

Biologie

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.

Bioingénierie de l&#39;homme Réseaux microvasculaires chez des souris immunodéficientes

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.

Expression et purification de la protéine transmembranaire de la fibrose kystique conductance Regulator dans 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.

Mesure de la conductance hydraulique foliaire et la conductance stomatique et de leurs réponses à d&#39;irradiance et de déshydratation en utilisant la méthode par évaporation 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.

Microscopie intravitale de la microcirculation dans le muscle de souris Cremaster pour l&#39;analyse des souches périphériques migration cellulaire

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;

Purification de la fibrose kystique régulateur de la conductance transmembranaire protéine exprimée dans<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.

Une nouvelle technique pour l&#39;analyse quantitative de la perte de cheveux chez la souris Utilisation de l&#39;analyse des niveaux de gris

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.

Concernant la conductance stomatique de feuilles traits fonctionnels

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.

Mesures de contractilité des muscles papillaires isolés sur pour l&#39;Investigation des inotropie cardiaque chez la souris

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.

Mesure de boucles de volume de pression chez la souris

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