Diese Arbeit wurde durch Mittel aus NIH gewährt HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) und ein Weltklasse-Universität Project (HJ) vom Ministerium für Wissenschaft, Technologie und Bildung von S. Korea unterstützt.

1. Teilweise Unterbindung der linken Arteria carotis <ol><li> Mäuse sind bei ~ Alter von 8 Wochen verwendet. Das Gewicht der Mäuse wird empfohlen, mindestens 18 Gramm betragen. </li><li> Sterilisieren der Instrumente für 30 Sekunden in bead Instrument Sterilisator und abkühlen lassen </li><li> Induce Anästhesie mit Ketamin (80mg/kg) und Xylazin (10mg/kg) intraperitoneal (ip) Injektion oder alternative Anästhesie durch IACUC genehmigt. Für ip-Injektion, injizieren Maus in der linken niedrigen Quadranten des Bauches. </li><li> Bewegen Sie die Maus in einer Wärmekammer, bis sie vollständig betäubt. Stellen Sie sicher, Narkosetiefe mit Reaktion bis zu den Zehen kneifen. </li><li> Bewegen Sie die Maus auf einer chirurgischen Stadium, in Rückenlage (Bauch nach oben) stellen. Kleben Sie die Vorderpfoten Handflächen nach oben und die Hinterpfoten allein auf der Bühne. </li><li> Bewerben liberal Menge Enthaarungsmittel (zB Nair), um den Hals zwischen dem Unterkiefer und dem Brustbein. Massieren Sie, bis alle Haare entfernt. </li><li> Reinigen Sie das Enthaarungsmittel und gelten großzügige Menge von Betadine zu enthaarten Bereich. </li><li> Bereiten Sie 1 Zoll lange Abschnitte 6:0-Seidennaht für Ligationen. Jede Maus benötigen Sie zwei Stück der Naht. </li><li> Mit einem scharfen kleinen Schere, einen 4-6mm vertikalen Schnitt in der Mitte des Halses. Sowohl die Haut und das darunterliegende Faszie müssen geschnitten werden. </li><li> Bewegen Sie den linken Ohrspeicheldrüse von der Mittellinie nach links und beginnen unverblümt Sezieren nur rechts von der Luftröhre. Sie sollten in der Lage, die pulsierende linke A. carotis leicht zu identifizieren. </li><li> Folgen Sie der Carotis kaudal bis zur Bifurkation zu finden. </li><li> Unverblümt sezieren der Bifurkation, die folgenden vier distalen zweigt die linke Arteria carotis communis freizulegen: Arteria carotis externa, Arteria carotis interna, A. occipitalis und A. thyreoidea superior. Diese Schiffe, vor allem überlegen Schilddrüse, kann brüchig. Seien Sie sehr vorsichtig mit Ihrem Dissektion. </li><li> Vernähen der A. carotis externa über dem oberen Schilddrüse mit 6-0 Seidenfaden. Um dies zu tun, Erster Durchlauf Pinzette unterhalb der Arterie und schnappen Sie sich einen Stück vorgeschnittenen Fäden mit Hilfe von Ihren anderen Pinzette. Ziehen Sie die Naht bis unter die Arterie passieren. Tie fest Vermeidung jeglicher umgebenden Gewebe in den Knoten gefangen. Wenn Sie binden unter dem Abheben des Arteria thyreoidea superior, durch das Ende der Prozedur, die Sie effektiv eine komplette Ligatur, die eine wesentlich andere Modell ist durchgeführt haben. </li><li> Vernähen der A. carotis interna und occipital Arterien mit einem Knoten mit der gleichen Technik. </li><li> Stellen Sie sicher, Position der Nähte und die Durchgängigkeit der Arteria thyreoidea superior. </li><li> Ungefähre der Haut und in der Nähe der Haut mit einem kleinen Betrag von Tissue-Mend. </li><li> Legen Mäuse in einer Wärmekammer bis zur Genesung. Wiederherstellung dauert in der Regel 30 bis 60 Minuten. </li><li> Eine einzelne Dosis von Buprenorphin (0.1mg/kg) sollten sofort verabreicht werden nach der Operation, wenn die Anästhesie nicht zur Verfügung stellt längerer Schmerzlinderung (zB inhaliert Isofluran) oder wenn das Tier in Not ist innerhalb der ersten 24 Stunden. Nach unserer Erfahrung bietet die Ketamin-Dosis für die Anästhesie verabreicht ausreichende Analgesie bei Operationen durch erfahrene Chirurgen durchgeführt wird und keine weitere Analgesie erforderlich ist. </li></ol> 2. Karotis-Ultraschall-Untersuchung <ol><li> Zur Validierung, ob die teilweise Ligation induzierten Fluss gestört, sollte carotis flow untersucht 1 Tag post-Ligation </li><li> Induce Narkose mit Isofluran - Narkoseeinleitung Box könnte bei Bedarf verwendet werden </li><li> Legen Sie narkotisierten Mäusen auf die Bildgebung der Bühne, Bauch nach oben </li><li> Band Arme und Beine, um EKG-Sensor in der Bildgebung der Bühne </li><li> Entlauben Hals durch liberale Anwendung von Nair (dieser Schritt ist nur erforderlich, wenn nicht bereits für die Ligation Verfahren erledigt) </li><li> Legen rektale Thermometer Temperatur aufzeichnen </li><li> Bewerben Echo-Gel auf den Hals </li><li> Start in B-Modus. </li><li> Imaging Flugzeug sollte 90 Grad (Knopf an der Sonde sollte nach der Nase der Maus Punkt) </li><li> Lower-Sonde auf Maus Hals, bis das Bild entsteht. </li><li> Manipulieren der Bühne links und rechts, bis Luftröhre (Mitte-line-Struktur) identifiziert </li><li> Identifizieren linken Arteria carotis communis </li><li> Tilt Bildgebung Bühne, so dass ein Winkel zwischen Bildebene und der Carotis erstellt. Je größer dieser Winkel ist, desto stärker ist das Doppler-Signal werden. </li><li> Legen PW-Doppler in der Mitte der linken Arteria carotis. </li><li> Verwenden Sie Winkelkorrektur nach Bedarf. </li><li> Wiederholen Sie die Messungen auf der rechten Arteria carotis. </li><li> Erfolgreiche partielle carotis Ligation wird in Gesamtreduktion in flow (~ 80-90%) in die linke A. carotis im Vergleich mit der rechten Seite durch, mit Umkehrung der Strömung in Richtung Aorta Einlass während der Diastole. </li><li> Sobald zufriedenstellende Bilder erhalten worden sind, schalten Sie Anästhesie, wischen echo off Gel Mäusen und Mäusen frei vom Band restraints. Legen Mäuse in erwärmt Erholung Kammer. </li></ol> 3. Intima RNA-Isolierung aus A. carotis <ol><li> Sacrifice Mäuse durch CO 2-Inhalation nach den institutionellen IACUC Protokoll </li><li> Kleben Sie die Pfoten auf einem Papiertuch. </li><li> Schneiden Sie die Haut der Maus aus dem Bauch nach oben des Thorax. </li><li> Öffnen der Bauchdecke unterhalb des Brustkorbs mit einer scharfen Schere. </li><li> Heben Sie das Brustbein mit einer Pinzette und schneiden das Zwerchfell, dann schneiden Sie den Brustkorb in das Herz aussetzen. </li><li> Schneiden Sie die Hohlvene mit einer Schere. </li><li> Pressure perfuse (120 mmHg) für 2 bis 3 min mit physiologischer Kochsalzlösung mit 10 Einheiten / mL Heparin durch den linken Ventrikel, bis die Lunge und Leber blass geworden. </li><li> Schneiden Sie die Haut des Halses und entfernen Sie alle Fett, Muskeln und Bindegewebe, bis die Halsschlagadern ausgesetzt sind. </li><li> Legen Sie die Maus unter einem Binokular. </li><li> Punktion ein Loch rechts unterhalb der Ligatur Standorten in der linken Halsschlagader zum zweiten Perfusion. </li><li> Pressure perfuse wieder für ~ 1 min mit physiologischer Kochsalzlösung mit 10 Einheiten / mL Heparin durch den linken Ventrikel, so dass die linke Halsschlagader gut durchgelesen. </li><li> Mit einer feinen Spitze Pinzette und kleine Feder Schere, vorsichtig den peri-adventitielle Gewebe um die Karotiden. Achten Sie darauf, nicht zu quetschen oder dehnen Sie die Carotiden in dieser Reinigungsstufe. </li><li> Schneiden Sie die linke Halsschlagader zwischen dem Aortenbogen und die Ligation-Punkten über dem Karotisgabel. </li><li> Schneiden Sie die rechte Halsschlagader zwischen der rechten A. subclavia Verzweigung und der Carotis-Bifurkation. </li><li> Übertragen Sie die Karotiden zu einer 35 mm Kulturschale mit eiskaltem HBSS. Wenn nötig, vorsichtig entfernen Sie alle verbleibenden peri-adventitielle Gewebe. </li><li> Bereiten Sie eine Insulin-Spritze (10.3 ml Spritze) mit einer 29g Nadel durch die Befüllung mit 150 pl QIAsol Lysepuffer (Qiagen) pro Halsschlagader. </li><li> Vorsichtig Spitze der Nadel in das eine Ende der Halsschlagader. </li><li> Halten Sie die Halsschlagader und die Spitze der Nadel mit einer Zange, schnell (~ 1 sec) spülen QIAsol Lysepuffer (150 ul) in ein 1,5 ml Röhrchen (Intima Eluat). </li><li> Spülen Sie die Carotis übrig (media + Adventitia) einmal in HBSS, steckte es in ein 1,5 ml-Tube und Snap-in flüssigem Stickstoff einfrieren. </li><li> Intima Eluat und die gefrorenen Reste werden dann für Intima oder Media + Adventitia RNA-Isolierung mit dem miRNeasy Mini Kit (QIAGEN) nach den Anweisungen des Herstellers verwendet. </li></ol> 4. Repräsentative Ergebnisse: Wir teilweise ligiert Maus nach links Halsschlagadern und Ultraschall-Untersuchung erfolgte 1 Tag nach dem Eingriff durchgeführt. Wie in Abbildung 1 dargestellt, reduziert erfolgreiche partielle Ligation Blutflussgeschwindigkeit und kehrt flow (gestörten) in der linken Arteria carotis communis während der Diastole. Teilweise Ligation von ApoE knockout (KO) Mäusen durch eine fettreiche Ernährung begleitet induziert robust Atherosklerose in der linken Arteria carotis communis (LCA), aber nicht in der kontralateralen rechten Carotis, um zwei Wochen (Abbildung 2). RNA aus der Intima gesammelt enthält endothelial Markergene wie PECAM-1, während frei von medialen glatten Muskelzellen Markergene wie a-SMA (Abbildung 3). <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Schematische Darstellung der A. carotis Anatomie und Ligationen: Drei Äste der linken Arteria carotis communis (LCA) [A. carotis externa (ECA), A. carotis interna (ICA), und A. occipitalis (OA)] wurden ligiert, während die Arteria thyreoidea superior (STA) zu öffnen. <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Abbildung 2" /> Abbildung 2: Die Ultraschall-Bilder zeigen Strömungsgeschwindigkeit Profile und zeigen, dass partielle Ligation Strömungsumkehr (durch Pfeile angedeutet) induziert in LCA während der Diastole. Flow in RCA bleibt unverändert nach der Ligation. <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Abbildung 3" /> Abbildung 3: Partielle Ligation und fettreichen Diät schnell eine Arteriosklerose in LCA von ApoE KO-Mäusen ApoE KO-Mäuse wurden teilweise unterbunden und fütterte die fettreiche Ernährung für 1 bis 3 Wochen.. Gefrierschnitte von LCA wurden mit Oil-Red-O gefärbt. <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Methode der Intima-RNA-Präparation. Intima RNA und medialen + Adventitia (m + a) RNA wurden aus scheinoperierten RCA und LCA in C57BL / 6 Mäusen erhalten. RNAs wurden durch semi-quantitative RT-PCR (A) und qPCR (B, C) ​​für PECAM-1 und α-SMA mit 18 Jahren als interne Kontrolle analysiert. Balkendiagramme sind Mittelwert ± 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>

In diesem Verfahren wird eine ordnungsgemäße Identifizierung aller Zweige der A. carotis einschließlich der A. carotis externa, A. carotis interna, A. occipitalis und A. thyreoidea superior unerlässlich. Es ist auch wichtig, um das Strömungsbild mit Hilfe von Ultraschall als bewiesen zu überprüfen. In unserem erfahrenen Händen ist unser Erfolg von Teil-Ligation Operationen auf dem Ultraschall-Untersuchungen von mehr als 500 Mäusen besser als 90%. Für die Karotis-Intima-RNA-Präparationen, ist es wichtig, die A...

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<td>Normal Saline (0.9% sodium chloride)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>2B1323</td>
<td>&nbsp;</td>
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<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>
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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>
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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

Trotz der bekannten engen Verbindung, hat einen direkten Beweis, der gestörten zur Atherogenese gefehlt. Vor kurzem haben wir eine modifizierte Version der Carotis partielle Ligation verwendeten Methoden [1,2] zeigen, dass es akut induziert niedrig und oszillatorische Strömungsverhältnisse, zwei wichtige Eigenschaften des gestörten, in der Maus Arteria carotis communis. Mit diesem Modell haben wir direkten Beweis dafür, dass gestörten Tat führt zu einer schnellen und robusten Atherosklerose Entwicklung in Apolipoprotein E-Knockout-Maus [3] zur Verfügung gestellt. Wir haben auch eine Methode entwickelt, von Endothel-RNA-Präparation mit hoher Reinheit aus dem Maus-Intima [3]. Mit dieser Maus-Modell und Methode fanden wir, dass partielle Ligation Ursachen endotheliale Dysfunktion in einer Woche, gefolgt von robusten und schnellen Atherombildung in zwei Wochen in einem hyperlipämischen Mausmodell mit Features von komplexen Läsionen Bildung wie intraplaque Neovaskularisation von vier Wochen. Diese schnelle<em&gt; In vivo</em&gt; Modell und die Endothel-RNA Präparationsmethode verwendet werden, um molekulare Mechanismen zugrunde liegenden Fluss-abhängige Regulation der vaskulären Biologie und Krankheiten zu bestimmen. Auch könnte es verwendet, um verschiedene therapeutische Interventionen gezielt endotheliale Dysfunktion und Atherosklerose in erheblich reduziert Studiendauer zu testen.

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

Dies beschreibt eine partielle carotis Ligation Chirurgie, die gestörte Strömungsverhältnisse und die anschließende Atherosklerose-Entwicklung (in zwei Wochen) führt mit intraplaque neo-Vaskularisation (in vier Wochen) in der Maus Arteria carotis communis. Wir beschreiben eine neuartige Methode der RNA-Isolierung aus der Intima, das hohe Reinheit endothelial RNA.

Ein Modell des Disturbed Flow-Atherosklerose in Maus Halsschlagader durch partielle Ligation und eine einfache Methode zur RNA-Isolierung aus Carotid Endothelium

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 Aufrufe.  Emory University. Dies beschreibt eine partielle carotis Ligation Chirurgie, die gestörte Strömungsverhältnisse und die anschließende Atherosklerose-Entwicklung (in zwei Wochen) führt mit intraplaque neo-Vaskularisation (in vier Wochen) in der Maus Arteria carotis communis. Wir beschreiben eine neuartige Methode der RNA-Isolierung aus der Intima, das hohe Reinheit endothelial RNA.

Serie: 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 einer Karotis Cuff für Triggering Shear-Stress-Induced Arteriosklerose bei Mäusen

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

Forschung

Medizin

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.

Trainieren eines Sophisticated mikrochirurgische Technik: Interposition der Vena jugularis externa Graft in der Arteria carotis communis bei Ratten

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.

Ein Mausmodell der Stentimplantation in der Arteria carotis für das Studium der Restenose

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.

Eine einfache Methode der Maus Lung Intubation

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.

Erwachsene Maus venöse Bluthochdruckmodell: A. carotis communis, um äußere Jugularvene 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.

Katheterisierung der Halsschlagader und Vena jugularis zur hämodynamischen Maßnahmen im Conscious Rattenmodell durchführen, Infusionen und Blutentnahme

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.

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

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.

Eisenchlorid-induzierte Thrombose Mausmodell für Halsschlagader und Mesentery Vessel

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 Gentransfer in die Kaninchen gemeinsame Halsschlagader Endothel

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.

Ein Kaninchen-Modell der dauerhafte Transgene Ausdruck in Halsschlagader, gemeinsame Halsschlagader 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, ...