Ce travail a été soutenu par un financement provenant de subventions des NIH HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) et une université de classe mondiale aux projets (HJ) du ministère de la Science, la Technologie et de l&#39;Education de S. Corée.

1. Ligature partielle de l&#39;artère carotide gauche <ol><li> Les souris sont utilisées à ~ 8 semaines d&#39;âge. Le poids de la souris est recommandé d&#39;avoir au moins 18 grammes. </li><li> Stériliser les instruments pour les 30 secondes dans le stérilisateur à l&#39;instrument de perles et laisser refroidir </li><li> Induire une anesthésie à la kétamine (80mg/kg) et de xylazine (10mg/kg) anesthésie intrapéritonéale (ip) par injection ou alternative approuvée par IACUC. Pour une injection ip, injecter de la souris dans le quadrant en bas à gauche de l&#39;abdomen. </li><li> Placez la souris dans une chambre de réchauffement jusqu&#39;à ce que complètement anesthésiés. Vérifiez la profondeur de l&#39;anesthésie avec réponse à pincement de l&#39;orteil. </li><li> Placez la souris sur une scène chirurgicale, en position couchée (côté ventre) position. Tape la patte avant-paumes vers le haut et la semelle pattes arrière vers le bas pour la scène. </li><li> Appliquez la quantité d&#39;agent dépilatoire libérale (par exemple Nair) au cou entre la mandibule et le sternum. Massez doucement jusqu&#39;à ce que tous les cheveux sont enlevés. </li><li> Nettoyez l&#39;agent à épiler et appliquer une quantité libérale de bétadine à la zone épilée. </li><li> Préparer 1 pouce de long sections de 6-0 suture de soie pour les ligatures. Chaque souris aura besoin de deux morceaux de fil de suture. </li><li> En utilisant une petite ciseaux pointus, faire une incision de 4 à 6 mm vertical dans le milieu du cou. Tant la peau et le fascia sous-jacents doivent être coupés. </li><li> Déplacer la glande parotide gauche de la ligne médiane vers la gauche et commencer carrément dissection juste à droite de la trachée. Vous devriez être capable d&#39;identifier la pulsation carotidienne commune gauche facilement. </li><li> Suivez la carotide commune caudalement jusqu&#39;à ce que vous localiser la bifurcation. </li><li> Carrément disséquer la bifurcation pour exposer les quatre branches distales de l&#39;artère carotide commune gauche: l&#39;artère carotide externe, l&#39;artère carotide interne, artère occipitale et l&#39;artère thyroïdienne supérieure. Ces navires, en particulier la thyroïde supérieure, peut être fragile. Soyez très prudent avec votre dissection. </li><li> Nouez la carotide externe au-dessus de la thyroïdienne supérieure en utilisant 6-0 suture de soie. Pour ce faire, passer une pince au-dessous de l&#39;artère et de saisir un morceau de fil de suture prédécoupés avec l&#39;aide de votre pince à d&#39;autres. Tirez doucement sur la suture de passer en dessous de l&#39;artère. Attachez solidement en évitant toute tissus environnants d&#39;être pris dans le noeud. Si vous attachez-dessous du décollage de l&#39;artère thyroïdienne supérieure, d&#39;ici la fin de la procédure vous sera effectivement avoir effectué une ligature complète qui est un modèle sensiblement différent. </li><li> Nouez les artères carotides internes et occipital avec un noeud en utilisant la même technique. </li><li> Vérifiez la position des points de suture et la perméabilité de l&#39;artère thyroïdienne supérieure. </li><li> Approximative de la peau et la peau à proximité en utilisant une petite quantité de tissu-Mend. </li><li> Souris Placer dans une chambre de réchauffement jusqu&#39;à la guérison. Le rétablissement prend généralement 30 à 60 minutes. </li><li> Une seule dose de buprénorphine (0.1mg/kg) doit être administré immédiatement après la chirurgie si l&#39;anesthésie utilisée ne fournit pas de soulagement de la douleur prolongée (ie. l&#39;isoflurane en inhalation) ou si l&#39;animal est en détresse dans les premières 24 heures. Dans notre expérience, la dose de kétamine administrés pour l&#39;anesthésie fournit une analgésie suffisante lorsque la chirurgie est effectuée par un chirurgien expérimenté et aucun analgésique supplémentaire est nécessaire. </li></ol> 2. Échographie carotidienne <ol><li> Pour valider si la ligature partielle induite perturbée débit, de débit carotidien doit être examiné un jour après la ligature </li><li> Induire une anesthésie avec l&#39;isoflurane - boîte induction anesthésique pourrait être utilisée si nécessaire </li><li> Placez la souris anesthésiée sur scène l&#39;imagerie, le côté du ventre jusqu&#39;à </li><li> Ruban bras et les jambes à l&#39;ECG du capteur sur scène l&#39;imagerie </li><li> Défolier le cou par une application libérale de Nair (cette étape n&#39;est nécessaire que si ce n&#39;est déjà fait pour la procédure ligature) </li><li> Insérer un thermomètre rectal pour enregistrer la température </li><li> Appliquez un gel Echo au cou </li><li> Démarrer en mode B. </li><li> Plan d&#39;imagerie doit être de 90 degrés (bouton sur la sonde doit pointer vers le nez de la souris) </li><li> Basse sonde sur le cou de la souris jusqu&#39;à l&#39;image est obtenue. </li><li> Manipuler étape de gauche et à droite jusqu&#39;à la trachée (mi-ligne de structure) est identifié </li><li> Identifier artère carotide commune gauche </li><li> Stade de l&#39;imagerie Tilt sorte qu&#39;un angle est créé entre le plan de l&#39;imagerie et la carotide. Plus cet angle, plus le signal doppler sera. </li><li> Placez impulsions Doppler au milieu de la carotide commune gauche. </li><li> Utiliser la correction d&#39;angle, au besoin. </li><li> Répéter les mesures sur la carotide commune droite. </li><li> Succès ligature carotidienne partielle entraînera une réduction globale des flux (~ 80-90%) dans la carotide commune gauche par rapport à la droite, avec inversion des flux vers l&#39;entrée aortique pendant la diastole. </li><li> Une fois les images satisfaisants ont été obtenus, éteignez l&#39;anesthésie, essuyez l&#39;écho de gel hors souris et souris sans res bandestraints. Placez la souris dans la chambre de récupération réchauffé. </li></ol> 3. Intima l&#39;ARN isolement de la carotide <ol><li> Sacrifice souris par inhalation de CO 2 selon le protocole institutionnel IACUC </li><li> Bande les pattes d&#39;une serviette en papier. </li><li> Coupez la peau de la souris de l&#39;abdomen vers le haut du thorax. </li><li> Ouvrez la paroi abdominale en dessous de la cage thoracique avec une paire de ciseaux. </li><li> Soulevez le sternum avec une pince et couper la membrane, puis couper la cage thoracique afin d&#39;exposer le cœur. </li><li> Couper la veine cave avec des ciseaux. </li><li> Perfuser pression (120 mmHg) pendant 2 à 3 min avec une solution saline normale contenant 10 unités / ml d&#39;héparine dans le ventricule gauche jusqu&#39;à ce que les poumons et le foie devient pâle. </li><li> Coupez la peau du cou et de retirer toute la graisse, les muscles et les tissus conjonctifs jusqu&#39;au artères carotides sont exposés. </li><li> Placez la souris sous un microscope à dissection. </li><li> Crevaison un trou juste au-dessous des sites ligature de la carotide gauche pour la seconde perfusion. </li><li> Pression perfuser à nouveau pour ~ 1 min avec une solution saline normale contenant 10 unités / ml d&#39;héparine dans le ventricule gauche, en veillant à la carotide gauche est bien lu. </li><li> En utilisant une pince à pointe fine et ciseaux petit ressort, retirer soigneusement les tissus péri-adventiciel entourant les carotides. Attention à ne pas presser ou étirer les carotides pendant cette étape de nettoyage. </li><li> Couper l&#39;artère carotide gauche entre la crosse de l&#39;aorte et les points ci-dessus ligature de la bifurcation carotidienne. </li><li> Couper l&#39;artère carotide droite entre le point artère sous-clavière droite de branchement et de la bifurcation carotidienne. </li><li> Transfert des carotides à une boîte de culture de 35 mm, contenant glacée HBSS. Si nécessaire, retirez soigneusement toute restantes péri-adventiciel tissus. </li><li> Préparer une seringue à insuline (seringue de 3 ml / 10) avec une aiguille 29g en remplissant avec 150 tampons QIAsol lyse ul (Qiagen) pour chaque carotide. </li><li> Insérez soigneusement la pointe de l&#39;aiguille à une extrémité de la carotide. </li><li> Tout en tenant la carotide et la pointe de l&#39;aiguille avec une pince, rapidement (~ 1 sec) rincer le tampon de lyse QIAsol (150 pl) dans un tube de 1.5ml (éluat intima). </li><li> Rincer les restes de la carotide (médias + adventice) une fois dans HBSS, le mettre dans un tube de 1,5 ml, et enclenchez-gel dans l&#39;azote liquide. </li><li> Intima éluat et les restes congelés sont ensuite utilisées pour l&#39;intima média ou de l&#39;isolement adventice + ARN en utilisant l&#39;miRNeasy mini kit (QIAGEN) selon les instructions du fabricant. </li></ol> 4. Les résultats représentatifs: Nous partiellement ligaturée gauche de la souris artères carotides et l&#39;échographie a été effectuée un jour après la procédure. Comme le montre la figure 1, le succès ligature partielle réduit la vitesse du flux sanguin et inverse de flux (flux perturbé) dans l&#39;artère carotide commune gauche durant la diastole. Ligature partielle de l&#39;ApoE KO (KO) accompagné d&#39;une souris régime riche en graisses provoque l&#39;athérosclérose robuste dans l&#39;artère carotide commune gauche (ACV), mais pas dans la carotide droite controlatéral, par deux semaines (figure 2). ARN recueillies auprès de l&#39;intima carotidienne contient des gènes marqueurs endothéliaux tels que PECAM-1, alors libre de gènes médial du muscle lisse marqueur de cellule comme un SMA-(figure 3). <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Figure 1" /> Figure 1: Schéma de l&#39;anatomie carotides et des ligatures: Trois branches de l&#39;artère carotide commune gauche (ACV) [artère carotide externe (CEA), l&#39;artère carotide interne (ACI), et l&#39;artère occipitale (OA)] ont été ligaturés, tout en laissant l&#39;artère thyroïdienne supérieure (STA) ouvert. <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Figure 2" /> Figure 2: Les images de l&#39;échographie montre des profils de vitesse de débit et de révéler que la ligature partielle induit inversion de flux (indiquées par des flèches) dans l&#39;ACV pendant la diastole. Débit en RCA reste inchangé après la ligature. <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Figure 3" /> Figure 3: ligature partielle et à haute teneur en graisses induit rapidement l&#39;athérosclérose chez des souris KO LCA ApoE ApoE souris KO ont été partiellement ligaturée et nourris avec le régime riche en graisses pendant 1 à 3 semaines.. Des sections congelées de LCA ont été colorés avec l&#39;huile rouge-O. <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Figure 4" /> Figure 4. Méthode de préparation de l&#39;ARN intimale. Intimale et médiale ARN + adventiciel (m + a) ARN ont été obtenus à partir opération fictive RCA et dans la LCA souris C57BL / 6. ARN ont été analysés par des semi-RT-PCR quantitative (A) et qPCR (B, C) ​​pour PECAM-1 et α-SMA utilisant 18 comme contrôle interne. Les graphiques à barres sont des moyennes ± SEM, n = 3.

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li></ol>

Dans cette procédure, l&#39;identification correcte de toutes les branches de la carotide commune, y compris la carotide externe, la carotide interne, artère occipitale et l&#39;artère thyroïdienne supérieure est essentielle. Il est également important de vérifier la configuration de l&#39;écoulement en utilisant des ultrasons comme l&#39;a démontré. Dans nos mains expérimentés, notre taux de succès des chirurgies ligature partielle sur la base des études de l&#39;échographie de plus de 500 souris est sup...

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<td>Normal Saline (0.9% sodium chloride)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>2B1323</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe (3/10 ml 29g syringe)</td>
<td>&nbsp;</td>
<td>Tyco Health care</td>
<td>8881600145</td>
<td>&nbsp;</td>
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<td>HBSS</td>
<td>&nbsp;</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>21-023</td>
<td>&nbsp;</td>
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<td>miRNeasy mini kit (including, QIAzol)</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>217004</td>
<td>&nbsp;</td>
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<td>Vevo 770 High-Resolution Micro-Imaging System</td>
<td>&nbsp;</td>
<td>Visualsonics</td>
<td>a.Visualsonics 770 system b.Imaging stage c.Vevo integrated rail system d.RMV scan head for mice</td>
<td>or similar system</td>
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<td>6-0 Silk Suture, sterile</td>
<td>&nbsp;</td>
<td>Covidien</td>
<td>s-1172 c2</td>
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<td>Small scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5675</td>
<td>or similar</td>
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<td>Dissecting Forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5005</td>
<td>or similar</td>
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<td>Tissue Mend II</td>
<td>&nbsp;</td>
<td>Webster Vet Supply</td>
<td>07-856-7946</td>
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a model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of rna isolation from carotid endothelium

Malgré l&#39;association connue près, les preuves directes reliant écoulement perturbé à l&#39;athérogenèse a fait défaut. Nous avons récemment utilisé une version modifiée de la carotide méthodes ligature partielle [1,2] pour montrer qu&#39;il induit gravement les conditions de faible débit et oscillatoire, deux caractéristiques essentielles du flux perturbé, chez la souris l&#39;artère carotide commune. En utilisant ce modèle, nous avons fourni des preuves directes qui en découlent perturbée conduit en effet au développement rapide et robuste athérosclérose chez la souris knockout apolipoprotéine E [3]. Nous avons également développé une méthode de préparation de l&#39;ARN endothéliales avec une grande pureté de l&#39;intima carotidienne la souris [3]. L&#39;utilisation de ce modèle de souris et de la méthode, nous avons constaté que la ligature partielle entraîne une dysfonction endothéliale en une semaine, suivie de la formation d&#39;athérome robuste et rapide de deux semaines dans un modèle de souris hyperlipidémiques ainsi que des fonctions de formation de lésions complexes tels que la néovascularisation intraplaque de quatre semaines. Ce rapide<em&gt; In vivo</em&gt; Modèle et la méthode de préparation endothéliales ARN pourrait être utilisée pour déterminer les mécanismes moléculaires sous-jacents des flux régulation dépendante de la biologie vasculaire et les maladies. Aussi, il pourrait être utilisé pour tester différentes interventions thérapeutiques ciblant la dysfonction endothéliale et l&#39;athérosclérose de la durée d&#39;étude considérablement réduit.

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

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

Ceci décrit une ligature partielle carotide, ce qui provoque des conditions d&#39;écoulement perturbé et développement de l&#39;athérosclérose ultérieure (dans deux semaines) avec intraplaque néo-vascularisation (en quatre semaines) chez la souris l&#39;artère carotide commune. Nous décrivons également une nouvelle méthode d&#39;isolement d&#39;ARN à partir de l&#39;intima carotidienne, fournissant ARN haute pureté endothéliales.

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 ...

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K vues.  Emory University. Ceci décrit une ligature partielle carotide, ce qui provoque des conditions d'écoulement perturbé et développement de l'athérosclérose ultérieure (dans deux semaines) avec intraplaque néo-vascularisation (en quatre semaines) chez la souris l'artère carotide commune. Nous décrivons également une nouvelle méthode d'isolement d'ARN à partir de l'intima carotidienne, fournissant ARN haute pureté endothéliales.

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Implantation d&#39;une manchette carotidienne pour le déclenchement de cisaillement-stress athérosclérose induite chez la souris

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Recherche

Médecine

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Former une technique microchirurgicale sophistiquée: Interposition de greffe veine jugulaire externe dans l&#39;artère carotide commune chez les rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Une méthode simple d&#39;intubation poumon de souris

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

Cathétérisme de l&#39;artère carotide et Veine jugulaire faire pour effectuer des mesures hémodynamiques, Infusions et prélèvement sanguin dans un modèle de rat Conscients

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusions de l&#39;artère carotide pour pharmacocinétiques et pharmacodynamiques Analyse des taxanes chez la souris

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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.

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

In Vivo Transfert de gènes à l’endothélium de l’artère carotide commune lapin

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Un modèle de lapin d’Expression du transgène durables dans la veine jugulaire à des greffes d’Interposition artère carotide commune

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...