The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

NOTE: Toutes les procédures liées aux animaux décrits dans le présent protocole ont été préalablement approuvé par le soin et l&#39;utilisation institutionnelle animaux Comité Duke University (DUIACUC). 1. Culture cellulaire et cancer Injection <ol><li> Cultiver des cellules cancéreuses métastatiques marquées par fluorescence (par exemple des cellules de cancer du sein MDAMB231-GFP humaines, don du Dr. Patricia Steeg, NCI, et les cellules de sarcome de la souris YFP-étiquetés, don du Dr. David Kirsch, Duke University Medical Center, Département de radio-oncologie) dans un milieu de culture approprié (milieu de Eagle modifié par exemple par Dulbecco (DMEM) avec 10% de sérum bovin fœtal et 1% de pénicilline / streptavidine) à 37 ° C jusqu&#39;à environ 90% de confluence. </li><li> Trypsiniser cellules, les laver deux fois avec du PBS, compter, et ensuite les injecter dans les veines de la queue de 10 semaines vieux rats nus féminins de l&#39;isoflurane anesthésiés à 5 millions de cellules par animal, en utilisant une seringue avec une aiguille 27 G. Anesthes niveau chirurgicalesia est vérifiée par l&#39;absence de réaction de pincement de l&#39;orteil. </li></ol> 2. Suivi des métastases Utilisation microCT <ol><li> Examiner les rats une fois par semaine en utilisant un micro-CT / micro-irradiateur, pour détecter la présence de métastases au-dessus d&#39;environ 2 mm de diamètre de diamètre. Le micro-CT est commandé comme décrit précédemment 12. <ol><li> Rats soumis à 3% d&#39;isoflurane anesthésie préalable à l&#39;imagerie. Confirmez anesthésie profonde par pincement de l&#39;orteil. </li><li> Après le début de l&#39;anesthésie, transférer rapidement des rats au berceau de l&#39;imagerie dans la chambre de l&#39;imagerie et de se connecter via un nez cône à un mélange isoflurane-air à 2,5-3%. Ajuster la position du rat dans le berceau de façon que son thorax est positionné dans le faisceau de photons de la microCT scanner, en utilisant les commandes de position externes et délimiteur de laser sur le support de formation d&#39;image. Se assurer que la porte de la chambre d&#39;imagerie est fermé, pour protéger l&#39;enquêteur des rayons gamma. </li><li> Contrôler la position de l&#39;animal en utilisant à nouveaula caméra vidéo couleur. Effectuez une basse résolution test d&#39;imagerie CT terme, et d&#39;utiliser l&#39;image résultante pour régler le champ de vision aux dimensions xyz de la cavité thoracique. </li><li> Image du thorax de rat en utilisant un filtre 2 mm Al à 40 kV, 2,5 mA, et 0,008 voxel au 7 FPS et le réexpédier vers sa cage. Imagerie d&#39;un animal devrait prendre pas plus de 15 ou 20 min. Ne retournez pas un animal qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à guérison complète. Ne pas laisser un animal sans surveillance tant qu&#39;il a repris conscience suffisante pour maintenir décubitus sternal. </li><li> Confirmez métastases par l&#39;apparition d&#39;objets relativement radio-opaque qui ne peut être expliqué par des vaisseaux sanguins intrathoraciques (figure 1A) </li></ol></li></ol> Chirurgie 3. Chambre Fenêtre <ol><li> Anesthésie, les signes vitaux et veine de la queue cathétérisme <ol><li> Sélectionnez animaux avec la présence de la maladie métastatique. Injecter animal avec un d intrapéritonéaleose de 50 mg / kg de pentobarbital. Confirmez niveau anesthésie chirurgicale par pincement de l&#39;orteil avant de procéder. Note: protocoles d&#39;anesthésie doivent être adaptées à la configuration expérimentale respective. Pentobarbital a été choisi ici comme un anesthésique à action prolongée, afin d&#39;induire anesthésie profonde pendant de longues procédures, tout en offrant la possibilité de re-dosage facile. Toutefois, la perte d&#39;animaux à un surdosage est un problème commun avec anesthésie au pentobarbital. Une autre option qui préserve réflexes autonomes à un degré plus grand que le pentobarbital est la kétamine en combinaison avec des sédatifs tels que la xylazine ou médétomidine, qui permet cependant uniquement pour un cycle de ré-dosage unique. </li><li> Raser les animaux sur le côté du corps qui a la maladie métastatique, et dans la région du cou en utilisant une tondeuse. Essuyer tout en restant cheveux dénoués de la peau. Après les cheveux dénoués est retirée, appliquer une pommade vétérinaire pour les yeux, pour les empêcher de se dessécher. REMARQUE: rats nus athymiques peuvent avoir les cheveux résiduel qui requiélimination res avant de procéder à des interventions chirurgicales. Il est très important d&#39;enlever tous les cheveux soigneusement, car il peut interférer avec les procédures chirurgicales et d&#39;imagerie. </li><li> Fixer l&#39;animal dans une position couchée sur une plaque métallique qui est placé sur un coussin chauffant l&#39;eau C 37 ° distribué. Les extrémités avant et arrière sont fixés sur la plaque avec du ruban adhésif. NOTE: Il est utile de contrôler et d&#39;enregistrer les signes vitaux, tels que la fréquence cardiaque et l&#39;oxygénation artérielle du sang en utilisant un oxymètre de pouls, tout au long des procédures chirurgicales et expérimentales. </li></ol></li><li> Intubation trachéale <ol><li> Afin de placer un cathéter pour la ventilation de l&#39;animal, d&#39;abord faire une incision transversale du col de la peau, suivie de la séparation médiane ventrale de la musculature longitudinale de la trachée. </li><li> L&#39;utilisation répétée des mesures ouverture à la fermeture avec une forte pince pour créer un passage pour le fil de suture à travers la face dorsale de la trachée. </li><li> Faire une petite incision dans la trachée sur til face ventrale, pas plus que semi-circulaire, approximativement entre le deuxième et le troisième anneau trachéal. Laisser un temps suffisamment long portion de la trachée exposée sur la face dorsale, afin de permettre la fixation de la sonde trachéale. </li><li> Insérez un mm &quot;Y&quot; canule trachéale de 2,5 à 3,0 dans la trachée, et serrer avec une suture 4-0 monofilament. Se assurer que la canule est reliée à un ventilateur de pression cyclée avec une bouteille reliée au conduit d&#39;expiration qui est rempli de 6 cm d&#39;eau, pour maintenir la pression pulmonaire positive. Flot de gaz devrait être de 100% d&#39;oxygène, à moins expérimentalement souhaité autrement. </li><li> Insérer un cathéter avec une aiguille de 25 à 27 G, et rempli de solution saline héparinée dans l&#39;une des veines de la queue du rat, et fixer en place avec du ruban adhésif. REMARQUE: Veiller à la perméabilité du cathéter veine de la queue toute la procédure en injectant plusieurs reprises une petite quantité de sérum physiologique hépariné dans la veine de la queue. En outre, l&#39;intubation oro-trachéale, ce est à dire la direction d&#39;un trachéaletube par la bouche de l&#39;animal anesthésié et au-delà du larynx dans la trachée, est une alternative possible à la procédure de trachéotomie qui est décrit ici. Cependant, cette méthode nécessite une formation spéciale et de l&#39;expérience, pour éviter d&#39;endommager la trachée, et aussi pour empêcher l&#39;cathétérisme accidentelle de l&#39;œsophage. </li></ol></li><li> Application de la fenêtre pulmonaire <ol><li> Retirer la peau du côté de la poitrine où se trouve la maladie métastatique, en créant une incision, puis détacher la peau en utilisant une dissection mousse. Remarque: La peau peut être excisé et retiré après détachement </li><li> Passez en disséquant les deux couches de muscles recouvrant (pectoral, dentelé et grand dorsal), mais en laissant les muscles intercostaux intacte. Créer une perforation dans la cavité thoracique d&#39;environ 1,5 cm de diamètre, en enlevant des parties de typiquement deux nervures adjacentes. Idéalement, localiser la perforation dans la région de la sixième et de la septième ribs. </li><li> Ostéotomie: <ol><li> Pour minimiser les saignements et les dommages à la surface du poumon, tenir fermement la nervure à couper avec une pince chirurgicale dentées pendant la coupe. Avec des ciseaux chirurgicaux, couper la partie médiane de la première nervure, à un angle d&#39;environ 45 °, laissant le côté pointu de l&#39;os de côte reste pointant extérieur. </li><li> Par la suite, couper la face latérale de l&#39;os de la nervure d&#39;une manière similaire, en laissant à nouveau la partie pointue de l&#39;os de la nervure dirigée vers l&#39;extérieur, pour éviter d&#39;endommager la surface du poumon. </li><li> Répétez la procédure pour la nervure adjacente, puis couper les muscles intercostaux et retirer la pièce excisée. Pendant cette procédure, maintenir la pression de poumon de façon que l&#39;interaction mécanique entre la surface du poumon et la cage thoracique est minimisée. Pour ce faire, en régulant la pression appropriée de l&#39;inspiration sur le ventilateur. </li></ol></li><li> Insertion de la fenêtre: <ol><li> Insérer une fenêtre de poumon sur mesure, composé d&#39;une lamelle qui estrelié à une prise en plexiglas (figure 1B). Fixer la fenêtre à la douille par collage, ou par l&#39;application d&#39;une très petite quantité de graisse à vide. Insérez la fenêtre de façon que les métastases de surface sont situés à proximité du centre de la fenêtre. Si nécessaire, ajuster le trou inséré pour amener le micrométastases au centre de la fenêtre en agrandissant légèrement le trou sur le côté respectif. REMARQUE: maladie métastatique sur la surface pleurale peut être identifié en tant que points blancs clairement reconnaissables ou des zones à l&#39;intérieur des parties autrement à rose saumon couleur saines de la surface du poumon qui apparaissent principalement le long des fissures. Bien micrométastases peuvent se produire sur d&#39;autres secteurs de l&#39;extérieur pleural, les lignées cellulaires étudiées présentent presque toujours surfaçage micrométastases dans la zone perforée, une fois que la maladie métastatique peut être radiologiquement détecté. </li><li> Après l&#39;insertion de la douille dans la perforation, et la création d&#39;un contact direct avec la plèvre viscérale de la lUng, suturer les bords du cadre de la fenêtre au muscle intercostal environnant, en utilisant 4,0 suture monofilament (figure 1C). Utilisez une légère augmentation de la pression de l&#39;inspiration sur le ventilateur pour aider air résiduel de se échapper et pour créer un joint. Note: Le taux de rats de la respiration peut varier considérablement, en fonction du statut de l&#39;anesthésie, état ​​d&#39;excitation ou d&#39;anxiété, concentration en oxygène de l&#39;air inspiré (FiO2), etc. Il est recommandé de régler le taux de respiration entre 70 et 90 bpm. La pression de l&#39;inspiration devrait être réglé avec prudence et ne pas être réglé à plus de env. 8 cm H 2 O (0,6 mm de Hg), pour éviter d&#39;endommager la surface du poumon. </li></ol></li><li> Placer l&#39;animal dans un dispositif de retenue conçu sur mesure qui est conçu pour éliminer le mouvement dans la direction Z (figure 1D) sur une plaque d&#39;acier qui est positionné sur une thermostatique (électricité) couverture chauffante, sous un microscope à fluorescence. Contrôler le corps de l&#39;animalla température rectale à l&#39;aide d&#39;une thermistance. Ajuster les vis du dispositif de retenue d&#39;animal, et la pression d&#39;inspiration sur le ventilateur pour obtenir un contrôle optimal de mouvement latéral. Remarque: la respiration naturelle chez les mammifères implique tous les trois directions de mouvement du poumon et l&#39;extension de la poitrine:, dorso-ventral et cranio-caudale bilatérale. Afin de préserver le mouvement de la respiration naturelle autant que possible, il est important de minimiser la compression dans la direction Z dans la mesure nécessaire. Parce que l&#39;appuie-Z-dimensionnelle a le potentiel d&#39;introduire des artefacts qui peuvent affecter la circulation sanguine et d&#39;autres paramètres, il est conseillé de garder conditions constante pendant série de mesures répétées chez le même animal. </li></ol></li></ol> 4. imagerie et mesure du débit sanguin Microcirculatory <ol><li> Recueillir les globules rouges par ponction cardiaque et les étiqueter avec Dil (1,1-3,3,3 -dioctadecyl = = perchlorate, 3 =-tétraméthyl indocarbocyanine), comme décrit précédemment &lt;sup&gt; 10. </li><li> Injecter 300 ul de globules rouges marqués dans la veine caudale du rat avant l&#39;opération de la chambre de fenêtre est fait, pour éviter les effets première passe d&#39;adhérence à la vitre en verre. Éliminer les bulles d&#39;air dans la seringue ou un cathéter, que l&#39;introduction de l&#39;air dans la veine va inhiber d&#39;autres injections. </li><li> Image du flux sanguin avec une caméra CCD de microscope à -40 ° C puce température de refroidissement, et d&#39;environ 100X résolution globale (ce est à dire avec un objectif de microscope 10X, 10X et pré-caméra oculaire). Utilisez standards jeux de filtres rhodamine / TRITC (excitation 450-490 nm, émission&gt; 515 nm). Enregistrer le taux de trame et pixel résolution réelle des séquences d&#39;images qui en résultent. Marquer au moins 200 (idéalement environ 300) images par pile, afin d&#39;assurer une analyse réussie de la vitesse d&#39;écoulement. </li><li> Reconstituer la perte de fluide dans l&#39;animal en injectant environ 1 ml de solution saline ip chaque h. REMARQUE: Les paramètres expérimentaux qui impliquent une intervention, par exempleun médicament qui modifie la vitesse d&#39;écoulement du sang dans une métastase de cancer pulmonaire, nécessite des mesures répétées de la vitesse d&#39;écoulement du sang dans le cancer métastatique pulmonaire. Pour ces expériences étendues, il est important de reconstituer l&#39;animal avec suffisamment de liquide. </li><li> Euthanasier les animaux en perfusion de 3 ml de 3N KCl dans la veine de la queue </li><li> Évaluer les piles d&#39;images en utilisant un, disponible publiquement algorithme publié ordinateur basé sur Matlab qui créera flux niveaux de gris de la vitesse et des cartes de couleur codé pour tous les flux de traces de sang 10,11. Évaluer la suite les images en niveaux de gris résultant en utilisant un logiciel d&#39;analyse d&#39;image commerciale ou accessibles au public, tels que Image J, après un seuil hors des valeurs qui indiquent aucun mouvement de globules, ce est à dire pas de système vasculaire active. </li></ol>

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Les auteurs ne ont rien à divulguer.

On présente un modèle qui est possible à l&#39;évolution de l&#39;image dans le débit sanguin microcirculatoire et d&#39;autres processus dynamiques dans des métastases pulmonaires de rat, en utilisant la microscopie intravitale et l&#39;analyse de la circulation sanguine calcul. Alors que d&#39;autres méthodes existent pour effectuer la microscopie sur les poumons exposés en cages thoraciques ouvertes de rongeurs, ce modèle est également le premier à l&#39;image des métastases pulmonaires par une perforatio...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

La vascularisation dans les tumeurs solides est connu pour différer considérablement de l&#39;approvisionnement en sang normale, montrant de plus grands degrés de tortuosité et les distances intervasculaires plus élevés 13. En conséquence, les pistes de la circulation sanguine dans le cancer du sein et de métastases pulmonaires sarcome expérimentales ont des formes irrégulières et de grandes lacunes intervasculaires (figure 2A, deux panneaux inférieurs) par rapport à la microcirc...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Mesure automatisée du flux sanguin Microcirculatory vitesse dans métastases pulmonaire des rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K vues.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Vidéo: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

La mesure in vivo de la surface de la couche de souris pulmonaire endothéliale

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Recherche

Médecine

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Mesures microscopie intravitale Vidéo de la rétine débit sanguin chez les souris

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Mesure automatisée de l&#39;emphysème pulmonaire et petites remodelage des voies aériennes dans la cigarette de fumée souris exposées

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

L&#39;augmentation de l&#39;artère pulmonaire pulsatile débit améliore l&#39;hypertension pulmonaire hypoxique chez les porcelets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Transcutanée Microcirculatory Imagerie en nouveau-nés prématurés

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Un modèle de Cardiac Remodeling Grâce à constriction de l&#39;aorte abdominale chez les rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

Enregistrement simultané de l’électrocardiographie et envahissantes sang pression mesure chez les Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Évaluation de la réserve de flux coronaire après la réperfusion d'ischémie myocardique chez les rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Test d’hémocompatibilité des implants de contact sanguin dans un modèle de boucle de flux imitant le flux sanguin humain

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.