Les auteurs tiennent à remercier le soutien technique de Joy Yao et le Dr Karen Alt, ainsi que le financement du NHMRC et NHF.

Toutes les expériences impliquant des animaux ont été approuvés par le Comité de l&#39;éducation Cité animale éthique (E / 1534/2015 / B) de la recherche médicale et Alfred. Toutes les manipulations chirurgicales ont été réalisées sous anesthésie générale et les animaux ne ressentent de la douleur à tout moment. Toutes les expériences décrites sont non-recouvrement. 1. Préparation <ol><li> Couper des bandes minces de papier filtre (1 mm x 2 mm). </li><li> Fraîchement préparer deux solutions de chlorure ferrique de 4% (p / v) et 6% (p / v) dilué dans de l&#39;eau désionisée. Préparer la solution de rhodamine 6G 0,3% en PBS, filtré à travers de 0,22 um. </li><li> Coupez un petit morceau (5 mm x 1 cm) à partir du plastique blanc de la seringue wrapper. </li></ol> 2. Formation Mesentery Artériole thrombus Observé par Microscopie intravitale <ol><li> Peser un 10-12 semaines C57BL / 6 souris de type sauvage et anesthésier en conséquence avec un mélange de kétamine (100 mg / kg) et de xylazine (10 mg / kg) si une injection intrapéritonéale. Surveiller la profondeur de l&#39;anesthésie par la réponse aux pieds, la queue et / ou pincement de la peau, la réactivité de pincement de la cornée et palpébrale, et l&#39;absence de mouvement des moustaches. Si nécessaire, injecter une seconde dose de kétamine (50 mg / kg) pour maintenir l&#39;anesthésie de l&#39;animal. Appliquer vétérinaire pommade sur les yeux pour prévenir la sécheresse tandis que sous anesthésie. Placez la souris dans une petite boîte de Pétri placée sur un coussin chauffant réglé à 37 ° C. NOTE: Bien que l&#39;injection IP peut entraîner des douleurs pour les animaux, la durée de ce malaise sera minime (moins de 3 secondes). La douleur et l&#39;inconfort associés aux injections seront minimisés grâce à l&#39;utilisation de personnel expérimenté et compétent et la taille de l&#39;aiguille appropriée (25 G). Après toutes les procédures, tous les euthanasier les animaux en utilisant une surdose de kétamine et de xylazine suivie par dislocation cervicale. </li><li> Effectuer une incision médiane abdominale d&#39;environ 3 cm dans la peau et découpez soigneusement le péritoine. </li><li> Placez la souris sur le côté de l&#39;animalplat ri, extérioriser doucement ses intestins et écartez avec précaution le mésentère avec 2 bourgeons de coton pour apporter un récipient approprié à la surface de la boîte de Pétri. Séchez correctement avec un essuie-glace délicate. Remarque: Afin de limiter le mouvement des vaisseaux mésentériques, la papaverine peut être utilisé pour inhiber le péristaltisme intestinal. </li><li> Faire tremper la queue de la souris dans l&#39;eau chaude pour dilater les vaisseaux et injecter 30 pi de la rhodamine 6G (0,3%) dans la veine de la queue de la souris avec une seringue de 29 G pour marquer les leucocytes et les plaquettes. </li><li> Placez la boîte de Pétri sous un microscope inversé et se concentrer sur l&#39;artériole choisie en utilisant le canal de champ lumineux. </li><li> Faire tremper une bande de papier filtre avec 6% (p / v) de fer (III) de chlorure et d&#39;appliquer le papier filtre sur l&#39;artériole avec deux pinces; le premier à tenir le papier filtre, le second d&#39;appuyer doucement sur la zone d&#39;intérêt. Observer la formation de thrombus dans la 10 première secondes après le dépôt du papier filtre. NOTE: Il est tout à fait commà blesser la microvascularisation entourée et la précision de la déposition du papier filtre et la pression en douceur est donc important de limiter ce problème. </li><li> Observer la formation de thrombus par microscopie à fluorescence (canal TRITC: excitation pic 557 nm, le pic d&#39;émission 576 nm), à travers le papier filtre. Respecter les leucocytes et les plaquettes circulantes qui ont pris la rhodamine 6G et leur agrégation dans le thrombus est donc facile à identifier. </li><li> Prenez le papier filtre au bout de 1 min d&#39;exposition au fer (III) et le chlorure de continuer à suivre la formation du thrombus. Lavez le récipient avec du PBS. </li><li> Observer et enregistrer la formation dynamique du thrombus mis en évidence avec l&#39;étiquetage des plaquettes et leucocytes avec la rhodamine 6G. Capturer les images et mesurer la taille du thrombus. Les images présentées ici ont été obtenues avec un microscope intravitale inversé, si un objectif 4X, dans le canal de fluorescence TRITC. </li><li> FA près toutes les procédures, l&#39;euthanasie de l&#39;animal en utilisant une surdose de kétamine et de xylazine, suivie par dislocation cervicale. </li></ol> 3. l&#39;artère carotide formation de thrombus évaluée par Circulation sanguine Mesure de vitesse <ol><li> Peser un homme âgé de 10 à 12 semaines C57BL / 6 et souris anesthésier en conséquence avec un mélange de kétamine (100 mg / kg) et de xylazine (10 mg / kg), même si l&#39;injection intrapéritonéale. Appliquer vétérinaire pommade sur les yeux pour prévenir la sécheresse tandis que sous anesthésie. </li><li> Fixer la souris sous un microscope opératoire en utilisant du ruban adhésif sur les jambes, sur un coussin chauffant réglé à 37 ° C. </li><li> Faire un petit oreiller sur un essuie-glace et du ruban adhésif sous la tête de la souris pour élever la tête légèrement. Utilisez une boucle de fil avec des pinces pour tirer le museau (utilisez dents supérieures). Cela permettra d&#39;exposer la région de l&#39;artère carotide pour un accès facile. </li><li> Effectuer un petit 5 mm incision profonde de la peau directement en dessous de la mâchoire, jusqu&#39;au sternum. </li><li> Disséquer la fascia et isoler un fragment soit de l&#39;artère carotide commune gauche ou à droite au dessus de la bifurcation. </li><li> Introduire délicatement pincettes en-entre l&#39;artère et le nerf de les séparer. Ne pas déranger le nerf courir près de l&#39;artère et éviter de toucher trop de l&#39;artère carotide en soit cause des dommages au navire. Isoler une section d&#39;un moins 5 mm de l&#39;artère. </li><li> Sécher la zone de l&#39;artère correctement avec essuie-glaces afin d&#39;éviter que tout liquide interfère avec le FeCl 3. </li><li> Mettez le petit morceau de plastique blanc dans la partie isolée de la carotide commune de sorte que le FeCl3 ne pas tremper dans les tissus environnants. A cet effet, utiliser une deuxième pince pour amener la pièce à la première puis, lentement, faites glisser le papier plastique sous l&#39;artère. </li><li> Faire tremper une feuille de papier-filtre avec 4% (p / v) ou 6% (p / v) de chlorure ferrique et le placer autour de l&#39;artère. </li><li> Après 3 min d&#39;exposition, enlever le papier filtre, rincer avec du PBS et sécher la zone wessuie vec. </li><li> Placer la sonde de débit Doppler autour de la cuve à la zone blessée et commencer à enregistrer les changements dans le flux. Dans la carotide commune de souris adultes en bonne santé, l&#39;écoulement est généralement autour de 1 ml / min. Attention! le contact de la sonde avec du chlorure ferrique endommager la sonde de façon tout contact doit être évitée. Les données présentées ici ont été obtenues avec un appareil de mesure de débit Transonic System Inc., TS420 Module périvasculaire équipé d&#39;une sonde de débit Doppler Nano 0,5 PBS. REMARQUE: La concentration du chlorure ferrique peut modifier la cinétique de formation d&#39;un thrombus résultant en différents temps d&#39;occlusion. Ainsi, une exposition à 6% (p / v) de chlorure ferrique donne une occlusion rapide que l&#39;exposition à 4% (p / v) de chlorure ferrique. </li><li> Après toutes les procédures, euthanasier tous les animaux en utilisant une surdose de kétamine et de xylazine suivie par dislocation cervicale et nettoyez soigneusement la sonde Doppler. </li></ol>

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The authors have nothing to disclose.

Le chlorure induite modèle de thrombose ferrique est un excellent outil de recherche. Comme le montre cette étude, il est extrêmement facile à mettre en œuvre et lorsqu&#39;il est utilisé en combinaison avec la microscopie intravitale ou Doppler débitmètre, il fournit une bonne surveillance en temps réel de la formation de thrombus. Réglage de la durée d&#39;exposition et la concentration de FeCl 3, il offre également la possibilité de produire soit thrombus occlusif ou non occlusif. <p class...

L&#39;étude des mécanismes impliqués dans le développement de la thrombose et de l&#39;évaluation de l&#39;efficacité des médicaments anti-thrombotiques nécessite bien établi des modèles animaux expérimentaux. Modèles animales importantes ont été les premiers à être utilisés car ils fournissent de grands navires plus semblable à l&#39;homme que les rongeurs 1. Cependant, le coût élevé, les grandes installations nécessaires et la difficulté à les manipuler génétiquement sont des inconvénients majeurs à leur utilisation et de grands animaux sont maintenant limités à des études précliniques fin une fois les tests préliminaires sur des rongeurs ont donné des résultats concluants 2. Avec une large disponibilité de souches transgéniques et knock-out et leur petite taille qui minimise la quantité de médicaments antithrombotiques requises pour les essais in vivo, les souris sont principalement utilisés pour la recherche sur la thrombose. Par conséquent, plusieurs modèles de troubles thrombotiques ont été développées chez des souris 3. Beaucoup de modèles de thrombose établies perturbent les intimune couche de la paroi du vaisseau, suivie par l&#39;exposition de la matrice extracellulaire sous endotheliales à l&#39;écoulement de sang induire la formation de caillots sanguins 4. Le thrombus peut résulter de l&#39;exposition du collagène qui déclenche l&#39;activation des plaquettes et / ou de l&#39;exposition du facteur tissulaire qui active la cascade de coagulation 5. Plusieurs techniques sont ensuite utilisées pour atteindre la blessure de cuve initial. Pierangeli et al. A développé un modèle de rupture mécanique avec un outil de microchirurgie sur la veine fémorale 6. Kikushi et al. Décrit un modèle qui consiste en l&#39;administration d&#39;un composé réactif de photo (rose Bengale) qui accumule dans la bicouche lipidique des cellules endothéliales, suivie par l&#39;excitation déterminée de la paroi du vaisseau d&#39;intérêt avec une lumière verte (540 nm) 7. La blessure peut également être induite par un court-éclairage à haute intensité de laser d&#39;impulsion 8. Une autre technique d&#39;abord mis en place sur l&#39;artère carotide de ratsconsiste en l&#39;application topique de chlorure ferrique (FeCl 3) 9. Dans ce cas, les résultats récipient de dénudation de radicaux libres générés par FeCl3 qui provoque la peroxydation lipidique et la destruction des 10 cellules endotheliales. La blessure induit l&#39;expression de plusieurs molécules d&#39;adhésion de déclenchement adhérence et l&#39;agrégation plaquettaire ainsi que le recrutement des leucocytes. Il a été démontré que les leucocytes, en particulier les neutrophiles jouent un rôle crucial dans l&#39;activation de la cascade de coagulation du sang conduisant à la thrombose 11. Cette méthode est bien adaptée à reproduire la cascade de coagulation; les enquêteurs doivent garder à l&#39;esprit que, dans ce modèle de souris, la thrombose est généralement induite dans vaisseaux sains alors que la thrombose chez les humains se produit principalement en ex malade. vaisseaux athérosclérotiques. Comme ce modèle est très simple à mettre en oeuvre et est également efficace chez les souris, il est maintenant le mode de thrombose principalement utilisél pour petit animal in vivo. En outre, cette technique offre la possibilité d&#39;induire la formation de thrombus dans une variété de vaisseaux. vaisseaux cibles peuvent être des artères ou des veines de grand diamètre (carotide, fémorale, la veine cave) ou de petit diamètre (mésentère, cremaster) 12-14. Plus récemment, il a également été utilisé sur l&#39;artère cérébrale moyenne proximale de développer un modèle d&#39;AVC 15. La formation de thrombose peut être directement observé par microscopie intravitale après marquage fluorescent de plaquettes et de leucocytes ou surveillé en mesurant la diminution du flux sanguin avec une sonde de température ou un 12,16,17 de la sonde Doppler. Plusieurs paramètres tels que le temps d&#39;occlusion, temps de formation de thrombus ou de la taille d&#39;un thrombus peuvent ensuite être étudiés. Les différences physiologiques entre les navires d&#39;une enquête suite à des variations importantes dans le thrombus obtenu. Par conséquent, les chercheurs sélectionner habituellement le vaisseau cible en fonction des paramètres qui les intéressent la pour mesurerre et / ou le réglage ils veulent enquêter sur la maladie. Typiquement, le modèle sur l&#39;artère carotide est plus pertinente pour la recherche sur l&#39;athérothrombose liée à l&#39;infarctus du myocarde ou accident vasculaire cérébral alors que les études sur la veine cave sont plus pertinentes pour la recherche sur la thrombose veineuse profonde. L&#39;accessibilité des différents vaisseaux détermine également la méthode utilisée pour mesurer la croissance de thrombus. Par exemple, les vaisseaux mésentériques sont faciles d&#39;accès qui rend ce modèle bien adapté pour l&#39;observation microscopique intravitale et l&#39;étude de la dynamique de la formation de thrombus. L&#39;artère carotide est moins accessible mais plus grand débit permettant des mesures de sang et de fournir un excellent modèle pour étudier la thrombose occlusive. Le modèle de thrombose induite de chlorure ferrique a fourni d&#39;énormes progrès dans la compréhension de cette pathologie. Il a été utilisé dans de nombreuses études portant sur ​​le rôle du facteur de von Willebrand à la formation de thrombose 18,19. Combiné avec les adaptations génétiquestechniques de fication, il a permis l&#39;identification de plusieurs gènes spécifiques impliqués dans les troubles thrombotiques. Lamrani et al. par exemple ont montré qu&#39;un knock-in du gène JAK2 V617F est associée à une formation accélérée de caillot instable 20. Zhang et al. Ont étudié l&#39;implication physiologique du récepteur P2Y12 plaquettaire et démontré que des souris transgéniques surexprimant spécifiquement ce récepteur dans les plaquettes seulement, ont montré une formation de thrombus plus rapide et plus stable dans l&#39;artère mésentérique blessé avec FeCl 3 21. Le rôle crucial de l&#39;activateur de type tissulaire du plasminogène (tPA) et l&#39;activateur du plasminogène de type urokinase (uPA) dans le processus de dégradation de la fibrine a également été étudié dans ce procédé 22. En outre, ce modèle fournit également un moyen simple et précis de tester les capacités fibrinolytiques de nombreux nouveaux médicaments in vivo. Par exemple, Wang et al. Ont utilisé ce modèle pour ee validation préclinique d&#39;un nouvel activateur du plasminogène recombinant dirigé contre les plaquettes activées 23. Cette méthode a également permis la validation de protéines thérapeutiques isolés de la salive des tiques, chauves-souris vampires, et les moustiques ou à partir du venin des serpents avec identification spécifique de la cible 24-27. Ces exemples montrent la souplesse du modèle de chlorure ferrique. Dans cet article, nous nous concentrons sur deux méthodes et étudions chlorure ferrique thrombose induite sur deux différents type de navire; vaisseau mésentérique et l&#39;artère carotide.

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			<td>Iron (III) chloride 40 % (w/v)</td>
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			<td>Rhodamine 6G</td>
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			<td>Inverted microscope&#160;</td>
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			<td>Digital black-and-white camera&#160;</td>
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			<td>Doppler flowmeter</td>
			<td>Transonic</td>
			<td>TS420</td>
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			<td>Nano-doppler flow probe</td>
			<td>Transonic</td>
			<td>0.5 PBS</td>
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			<td>Ketamine</td>
			<td>Hospira&#160;</td>
			<td>0409-2051-05</td>
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			<td>Xylazine (Rampun)</td>
			<td>Bayer</td>
			<td>75313&#160;</td>
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			<td>Petri dish</td>
			<td>Sarstedt</td>
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			<td>Insulin syringe (29 G)</td>
			<td>BD Ultra-Fine</td>
			<td>326103</td>
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			<td>Cotton tipped applicators</td>
			<td>BSN medical</td>
			<td>211827A</td>
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			<td>Dynek dysilk sutures</td>
			<td>Dynek Pty Ltd</td>
			<td>CS30100</td>
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			<td>Dulbecco&#39;s phosphate buffer saline (PBS)</td>
			<td>Gibco life technologies</td>
			<td>21600-069</td>
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			<td>Heating pad</td>
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ferric chloride-induced thrombosis mouse model on carotid artery and mesentery vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

La fluorescence intravitale observation au microscope du mésentère révélera l&#39;accumulation de Rhodamine 6G plaquettes et les leucocytes marqués le long de la paroi du vaisseau lésé par FeCl 3. La formation progressive d&#39;un thrombus partielle est surveillée dans un récipient de mésentère 200 um (figure 1). Un thrombus apparaît lentement et clairement identifiée après la première minute d&#39;exposition à FeCl 3 (figure 1, t = 60 s). 40 second...

Watch this Scientific Journal Video about Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel at JoVE.com

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Chlorure ferrique induite souris thrombose type sur l&#39;artère carotide et Mesentery navire

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

ferric-chloride-induced-thrombosis-mouse-model-on-carotid-artery

Research

JoVE Journal

Medicine

17.3K vues.  Baker IDI Heart and Diabetes Institute. The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Vidéo: Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Un modèle de Disturbed flux athérosclérose induite chez la souris artère carotide par ligature partielle et une méthode simple d&#39;isolement d&#39;ARN de l&#39;endothélium carotidienne

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Recherche

Médecine

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Modèle de souris complète stase de la veine cave inférieure thrombose

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modèle murin d&#39;occlusion artère cérébrale moyenne

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Un modèle murin d&#39;implantation d&#39;une endoprothèse dans l&#39;artère carotide pour l&#39;étude de la resténose

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Adulte souris veineuse Modèle d&#39;hypertension: l&#39;artère carotide commune à externe Veine jugulaire anastomose.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Vasculaire Balloon blessures et de l&#39;administration chez le rat Intraluminal artère carotide

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

A Rat carotidienne Balloon Modèle de lésion pour tester Therapeutics Rénovation Anti-vasculaires

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Ferriques murin Thrombose modèles induits par Chlorure

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contraste d’imagerie par résonance magnétique dans l’artère carotide commune de Rat

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Thrombose de l’artère carotide Canine induite par le chlorure ferrique : Un grand modèle Animal de lésion vasculaire

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...