Name: murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch
Uploaded: 2015-02-10T14:00:00.000+00:00
Duration: 7 min 5 s
Description: Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director&#8217;s New Innovator Grant (1DP2 OD008716-01). The authors would like to thank the services provided by the ICMB (Institute of Cellular and Molecular Biology) core facility and TherapeUTex at University of Texas at Austin.

HINWEIS: Alle in diesem Protokoll aufgeführten Methoden wurden von der Institutional Animal Care und Verwenden Ausschuss gebilligt wurde. 1. Vorbereitung der OP-Tisch <ol><li> Richten Sie das Heizkissen mit Umlauf warmem Wasser auf dem OP-Tisch, unter dem Dissektionsmikroskop. Legen Sie die Grundplatte aus Edelstahl auf dem Heizkissen. Legen Sie eine sterile saugfähigen Kissen auf der Grundplatte und Heizkissen. </li><li> Vereinbaren sterilen chirurgischen Instrumente auf einem angrenzenden sterilen saugfähigen Kissen. Sammeln zwei Paare von abgewinkelten spitzen Pinzette zwei Paare von abgewinkelten Zange drei Arterienklemmen, zwei Retraktoren Drähte, kleine chirurgische Schere, Mikroscheren, einer Angioplastie Draht (0,15 Zoll) Durchmesser und drei 6-Zoll-Segmente 6.0 Seidennaht . </li><li> Beugen Sie das runde Ende des Angioplastie-Draht, um die Krümmung der Femoralarterie entsprechen. Dadurch wird es leichter, das Kabel während der Operation voraus. </li></ol> 2. Herstellung vonMaus für Chirurgie <ol><li> Anesthetize eine Maus bei kontinuierlichem Einatmen von 2,5% Isofluran. Achten Sie darauf, den Status des Tieres während des gesamten Verfahrens überwachen. Führen Sie eine Prise Test der Maus den Fuß, um zu bestätigen, dass es vollständig betäubt wird. Stellen Sie sicher, das Tier bewegt sich nicht, wenn die Prise Test verabreicht wird. </li><li> Tragen Sie eine Schmiersalbe die Augen des Tieres, um ein Austrocknen zu verhindern. Sichern Sie das Tier in Rückenlage auf dem absorbierenden Kissen mit chirurgischen Band. Weiter Verwaltung 2,5% Isofluran via Nasenkegel. </li><li> Mit einer Enthaarungscreme, entfernen Sie das Fell auf dem Bein und Bauch auf der Mittellinie. Mit Wasser ausspülen und die Haut gründlich. Depilation eines Tages vor der Operation durchgeführt werden. Beachten Sie, dass übermäßige Behandlung mit der Creme kann zu Hautreizungen führen. </li><li> Unmittelbar vor der Operation, gelten PVP-Jod auf die enthaarte Bereich mit einem Wattestäbchen, um die Haut zu sterilisieren. Spülen Sie die enthaarte Haut mit 70% Ethanol und trockenmit einem sterilen Wattestäbchen. 3-mal wiederholen. </li><li> Verwalten einer präoperativen Dosis von 5 mg / kg von Carprofen subkutan injiziert wird. </li></ol> 3. Isolierung der Oberschenkelschlagader <ol><li> Mit kleinen chirurgischen Schere, einen gebogenen Schnitt in der Haut über die Oberschenkelarterie. Stumpf sezieren und zu sichern umgebenden Gewebe mit Retraktoren Magnet Aufroller Fixatoren die Femoralarterie zu lokalisieren. Befeuchten Sie das Gewebe in regelmäßigen Abständen mit Kochsalzlösung für die Bewässerung Nehmen Kochsalzlösung mit einem sterilen Wattestäbchen. </li><li> Isolieren Sie die Oberschenkelschlagader mit einer Pinzette. Trennen Sie vorsichtig die Nerven aus dem Gefäßbündel mit spitzen Pinzette. Vermeiden Punktion der Vene und nicht die Nerven schädigen. Schieben Sie den Nerven vom Bündel eine Anregung es. </li><li> Trennen Sie vorsichtig die Vena femoralis von der Femoralarterie, Lokalisierung des Oberschenkel Bifurkation. Der Bereich der Bifurkation ist besonders schwierig zu sezieren. </li><li> Posterior zur Bifurkation, Schleife 6,0 Seidenfaden unter der Oberschenkelarterie und sicher mit einer Gefäßklemme. Diese proximale Nahtmaterial verwendet werden, um den Blutfluss in der Arterie zu beschränken. Hinweis: Es gibt eine leichte Variation in der Bande bei der Durchführung der Ligationsmethode gegenüber der Kauterisation Methode (siehe Abbildung 1 und Abbildung 2). </li><li> Distal der Bifurkation, Schleife 6.0 Seidenfaden unter der Oberschenkelarterie und sicher mit einer Gefäßklemme. Diese distale Naht Hilfsmittel bei der Positionierung der Arterie. </li><li> Schleife zwei Nähte unter der Muskel Zweig der Arteria femoralis, vorge binden sie und mit einer Gefäßklemme. Denken Sie daran, um das Gewebe mit Kochsalzlösung befeuchten. Bei Durchführung der Kauterisation Verfahren Draht Verletzung, ist nur eine Schlaufennaht an der Muskelast notwendig. </li></ol> 4. Durchführung der Femoralarterie Draht Injury <ol><li> Blutfluss in die Oberschenkelarterie zu beschränken, indem der proximalen Naht. Leicht ziehen Sie den distalen Hemostan und Gefäßklemme Sicherung der Niederlassung der Website für die Arteriotomie zu entlarven. Ligation der Muskulatur Zweig durch das Binden des Nahtmaterials um ihn herum. HINWEIS: Die Aufhebung der Arterie nach oben wird effektiver beschränken Blutfluss als Ziehen horizontal allein. </li><li> Sever den kleinen Zweig mit dem Brenneisen zwischen den beiden Nähten. Mit Mikro-Schere, führen Sie eine Arteriotomie im Seitenzweig der Bifurkation. Verwenden einer Pinzette auf eine Rolle von chirurgischem Band ausgewogen, um die Mikroscheren stabilisieren. </li><li> Bestätigen Sie die Anwesenheit der Arteriotomie mit spitzen Pinzette. Heben Sie die Öffnung der Arteriotomie mit der Zange. Führen Sie das abgerundete Ende des Drahtes in die Arteriotomie mit einer Pinzette. Zur Vorwärtsbewegung des Drahtes zu erleichtern, fügen ein oder zwei Tropfen von Lidocain auf die Region unter Verwendung einer Spritze. </li><li> Wenn der Draht das proximale Naht erreicht, lassen Sie den Faden und so einstellen, dass sie die Weiterentwicklung des Drahtes nicht zu behindern. Führen Sie den Draht, bis es nicht weiter vorantreiben. Die Spitze the Draht sollte im Bereich des Leistenbandes zu stoppen. </li><li> Lassen Sie den Draht in die Oberschenkelarterie für eine Minute bleiben. Nach einer Minute zurückziehen und rückt den Draht in einem Sägebewegung zehnmal zu verletzen und entblößen das Endothel der A. femoralis. Für eine Verletzung von geringerer Schwere, verringern die Anzahl der Male der Draht in und aus der Arterie herausgezogen oder aus dem Draht in der Arterie für 1 min nur. </li><li> Fahren Sie den Draht langsam. Wenn das runde Ende des Drahtes hat die proximale Naht geführt, beschränken Fluss in der Arterie, indem die proximale Naht. Fahren Sie den Draht vollständig. </li></ol> 5. Ligation des Muskulös Niederlassung <ol><li> Ziehen Sie die verbleibenden Naht auf der Muskel Branche. Dies wird Blutung aus der Arteriotomie zu verhindern. Rückfluss zum Muskel Zweig, und bestätigen, dass das Blut nicht aus ihm austritt. Schneiden Sie die Enden der Fäden auf der Muskel Branche. </li></ol> 6. Alternative Methode: Lokalisierte Caumetrierung des Arteriotomie HINWEIS: Ein alternativer Ansatz ergriffen werden können, um die Ligation der Muskulatur Zweig vermieden werden und Gefäßzugang durch die größere Hauptoberschenkelarterie. <ol><li> Ausgehend von 4,1, beschränken Fluss zur Oberschenkelarterie durch Ziehen am proximalen Nahtmaterial. Ziehen Sie den distalen Naht und die Befestigung von Nahtmaterial die Muskelzweig leicht auf die Seite für die Arteriotomie (Abbildung 1 und Abbildung 2) aus. </li><li> Mit Mikro-Schere, führen Sie eine Arteriotomie am Verzweigungspunkt der Oberschenkelarterie und der Muskel Branche. Wenn der Einschnitt in der Seite der Arterie, es leichter zu ätzen sein können. Verwenden einer Pinzette auf eine Rolle von chirurgischem Band ausgewogen, um die Mikroscheren stabilisieren. </li><li> Um den Erfolg der Kauterisation zu testen, wiederherzustellen Fluss zur Oberschenkelarterie durch das Lösen der proximalen Nahtmaterial. Wenn die Blutung aus der Arteriotomie auftritt, wiederholen Sie den Brenneisen. Wenn die Brenneisen erfolgreich ist, wird der Blutfluss wieder werdengespeichert distal der Arteriotomie. </li><li> Führen Sie den Draht, und führen Sie den Draht Verletzungen, wie in Kapitel 4 beschrieben. </li><li> Weiter Einschränkung des Blutflusses in die Oberschenkelarterie. Erhitzen Sie mit einem spitzen Kauter mindestens 6 Zentimeter entfernt von der Maus. Da die Brenneisen Spitze abkühlt, gelten sie leicht Seite des Arteriotomie, um den Einschnitt zu schließen. </li><li> Um den Erfolg der Kauterisation zu testen, wiederherzustellen Fluss zur Oberschenkelarterie durch das Lösen der proximalen Nahtmaterial. Wenn die Blutung aus der Arteriotomie auftritt, wiederholen Sie den Brenneisen. Wenn die Brenneisen erfolgreich ist, wird der Blutfluss distal der Arteriotomie wiederhergestellt werden. Entfernen Sie die temporären proximalen und distalen Krawatten. </li></ol> 7. Implantation Perivaskuläre Drug Delivery-Patch <ol><li> Erstellung einer perivaskulären Medikamentenpflaster nach früheren Studien 11 oder durch Verwendung ähnlicher Verfahren beschrieben erzeugt werden. Blunt die Ecken der Sperrbereich der Arzneimittel-Einführungspflasters mit einer sterilen Schere </li><li> Legen Sie die Medikamentenabgabe patch auf die verletzten Oberschenkelarterie mit Blick auf die Arterie der Arzneimittel freisetzenden Seite. Nutzen Sie gegebenenfalls Pinzette, um die Position des Patches zu verbessern. </li></ol> 8. Wundverschluss und Wiederherstellung <ol><li> Schließen Sie die Wunde mit einem einfachen Knopfnaht, die aus quadratischen Knoten. Eine diskontinuierliche Naht verlängert Wundverschluss bei dem Tier versucht, um die Naht zu entfernen. </li><li> Schalten Sie die Anästhesie und entfernen Sie das Tier aus der Einrichtung. Lassen Sie das Tier auf die Erwärmung pad erholen. </li><li> Weiterhin der Maus die Erholung zu überwachen. Überprüfen Sie die Inzisionsstelle jeden Tag, um sicherzustellen, dass es geschlossen bleibt. Für die Behandlung von postoperativen Schmerzen, verwalten eine subkutane Injektion von 5 mg / kg von Carprofen jeder 12 Stunden nach der Operation und dann alle 12 Stunden für 2 Tage. Wenn der Schmerz geht weiter über die ersten 2 Tage, konsultieren Sie einen Tierarzt nach dem Weg zu weiteren Schmerzmittel. </li></ol> 9. Ernten Femoralarterien für die Histologie <ol><li> 28 Tage nach der Operation, führen Kohlendioxid Euthanasie auf der Maus. HINWEIS: Die entsprechende Strömungsgeschwindigkeit des Kohlendioxids sollte 10-30% des Kammer pro Minute zu verdrängen und in Abhängigkeit von der Größe der verwendeten Kammer variieren. Schneiden der Membran und Herzpunktion als sekundäre Verfahren Euthanasie durchgeführt werden. </li><li> Sichern Sie die Maus in Rückenlage mit chirurgischen Band. Einen Einschnitt in die Femoralarterie, wo die ersten chirurgischen Eingriff vorgenommen wurde. </li><li> Sowohl für die verletzte Arterie und der unverletzten Arterie aus dem kontralateralen Bein, unverblümt zu sezieren und zu sichern umgebenden Gewebe mit Retraktoren und magnetische Aufroller Fixatoren die Oberschenkelschlagader zu finden. Befeuchten Sie regelmäßig mit Kochsalzlösung für die Bewässerung der Gewebe. Übernehmen mit einer sterilen Kochsalzlösung Wattestäbchen. Achten Sie darauf, um die Arterie nicht beschädigen. </li><li> Nachdem die Oberschenkelarterie wurde von der Stelle des ursprünglichen Arteriotomie in die Bauchaorta isoliert, binden eine segment der Seidenfaden in der Nähe von der Original-Website der Arteriotomie. Dieser Faden wird dazu beitragen, die meisten distale Ende des Oberschenkelarterie und einfache Handhabung der Probe. </li><li> Mit Hilfe der Mikro-Schere Dissektion der Arteria femoralis zu herauszuschneiden. Stellen Sie eine Inzision distal der Naht. Machen die anderen Einschnitt auf der gegenüberliegenden Seite des Oberschenkelarterie, neben der Bauchaorta. </li><li> Übertragen Sie die ausgeschnittene Arterie zu einer Glas-Petrischale Kochsalzlösung. Präparieren Sie die Arterie weiter, um überschüssiges Bindegewebe oder Fett zu entfernen. Blut Entfernen Sie vorsichtig von den Lumen durch Spülen mit Kochsalzlösung. </li><li> Übertragen Sie die Arterie in eine Ampulle von 10% gepuffertem Formalin. Lagern Sie die Fläschchen bei 4 ° C unter leichtem Schütteln für 48 Stunden. </li><li> Übertragen der Festader zu 70% Ethanol, zu speichern, bis sie für die Histologie verarbeitet. </li><li> Betten Sie die Arterie in die Paraffinblocks und Abschnitt die Blöcke für die Färbung. </li><li> Führen histochemische und immunochemischen Färbung, um das Ausmaß der Schädigung und der Intima hyp beurteilenerplasia. HINWEIS: Für die repräsentative Ergebnisse, Hämatoxylin und Eosin verwendet wir, um die Kerne und die allgemeine Morphologie oder eine Movat des Penta Fleck sichtbar, um den elastischen Lamellen und andere arterielle Komponenten sichtbar zu machen. </li></ol>

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(40), (2010).</li><li>Kuhlmann, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+a+carotid+cuff+for+triggering+shear-stress+induced+atherosclerosis+in+mice.">Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.</a> J Vis Exp. (59), (2012).</li><li>Carmeliet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+wound+healing+and+neointima+formation+induced+by+perivascular+electric+injury+in+mice.">Vascular wound healing and neointima formation induced by perivascular electric injury in mice.</a> Am J Pathol. 150 (2), 761-776 (1997).</li></ol>

We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire inju...

Arterial injury and inflammation caused by angioplasty and stent implantation can induce neointimal hyperplasia that contributes to the thickening of the arterial wall, a process known as restenosis.1,2 The formation of restenosis is major mode of failure for interventions such as angioplasty and stenting with bare metal stents.3 Due to recent concerns with the inhibition of vascular healing in arteries treated with drug eluting stents, there is also a need to find compounds that can inhibit restenosis while maintaining vascular healing and re-endothelialization.4-7 In addition, while stents have had success in the coronary vasculature, percutaneous interventions of all types in the peripheral arteries continue to fail at a higher rate due to restenosis.8-10 Mouse models of surgical interventions allow the use of powerful genetic manipulations that can provide mechanistic insight into the mechanisms underlying the failure of clinical therapies and can provide an initial test bed for compounds to inhibit intimal hyperplasia. 
Here, we describe a mouse model of vascular injury that allows the testing of therapeutic compounds to inhibit neointimal hyperplasia and assess whether their effects on re-endothelialization following endothelial denudation. A key challenge in executing vascular injury in mice is the technical skill needed to obtain vascular access and to restore flow to the injured artery following the wire injury. For this reason, simple arterial ligation models have been used to study neointimal hyperplasia in mice that do not require endovascular manipulations but are easier to implement.11 However, this type of surgical model differs substantially from the mechanical and biological aspects of a percutaneous intervention, lacking key aspects including arterial wall stretch, endothelial denudation and luminal blood flow following injury. 
We present two methods for obtaining and closing vascular access for wire injury of the femoral artery in mice. The first technique is the conventional method described by several groups previously and uses vascular access through a side branch of the femoral artery.12-14 This method requires older, larger mice and more surgical skill to implement the endoluminal access through the smaller artery. It also requires the ligation of the muscular branch of the femoral artery following the procedure. The second method we describe uses an arteriotomy in the branch point of the main and side branch and thereby allows for a larger access to the artery for performing wire injury. In this method, the arteriotomy is closed using controlled local cauterization that leaves both branches with blood flow following the procedure. The conventional method is applicable to mice of at least 20 weeks of age while the alternative method can be used in mice of at least 15 weeks of age. In both methods, the wire creates arterial stretch and abrasion leading to injury and endothelial denudation. Following either procedure a perivascular drug delivery patch can be implanted that allows the delivery of compounds to alter the response to injury. The use of the drug delivery patch allows mice to be used as a test bed for new compounds to inhibit restenosis through perivascular therapies.15,16

<table><tbody><tr><td>Straight spring wire, 0.15&amp;rdquo; diameter</td><td>Cook</td><td>G02426</td><td></td></tr><tr><td>High Temperature cautery</td><td>Bovie Medical Corp.</td><td>HIT1</td><td></td></tr><tr><td>High-temperature fine tip for cautery</td><td>Bovie Medical Corp.</td><td>H101</td><td></td></tr><tr><td>Micro-scissors</td><td>Fine Science Tools</td><td>15000-13</td><td>For performance of arteriotomy</td></tr><tr><td>Angled fine-tipped forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>For blunt dissection of vascular bundle</td></tr><tr><td>Angled forceps</td><td>Roboz</td><td>RS-5069</td><td>For clearing tissues</td></tr><tr><td>Surgical scissors</td><td>Roboz</td><td>RS-5840</td><td>For cutting skin</td></tr><tr><td>Retractor</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Retractor wire</td><td>Fine Science Tools</td><td>18200-05</td><td>Attached to retractor</td></tr><tr><td>Base plate</td><td>Fine Science Tools</td><td>18200-03</td><td>For use with retractor</td></tr><tr><td>Magnetic retractor fixator</td><td>Fine Science Tools</td><td>18200-01</td><td></td></tr><tr><td>Needle Holder</td><td>Roboz</td><td>RS-7822</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Biomedical Research Instruments, Inc.</td><td>34-1000</td><td></td></tr><tr><td>Dissecting microscope</td><td>Meiji Techno</td><td>EMZ-5TR</td><td></td></tr><tr><td>Microscope light source</td><td>Meiji Techno</td><td>FT191</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-500</td><td>For maintaining mouse body temperature</td></tr><tr><td>Reusable heating pad</td><td>Gaymar</td><td>TP-R 22G</td><td>For maintaining mouse body temperature</td></tr><tr><td>Lidocaine</td><td>Various</td><td></td><td></td></tr><tr><td>4.0 Vicryl suture with half circle needle</td><td>Ethicon</td><td>J494G</td><td>For post-surgical wound closure</td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Puritan</td><td>25-806-2WC</td><td>For application of depilatory cream and absorbing fluids</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Various</td><td></td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>6.0 braided silk suture</td><td>Teleflex Medical</td><td>4-S</td><td>For isolation of femoral artery during surgery</td></tr><tr><td>0.9% sodium chloride</td><td>Various</td><td></td><td>For irrigating tissues</td></tr><tr><td>Gel eye lubricant</td><td>Various</td><td></td><td></td></tr><tr><td>Glass Petri dish</td><td>Pyrex</td><td>3160-60</td><td>For femoral artery harvest</td></tr><tr><td>10% buffered formalin</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>Bouin&#039;s fluid</td><td>Electron Microscopy Sciences</td><td></td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcian blue, 1%</td><td>Electron Microscopy Sciences</td><td>26385-01</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alkaline alcohol</td><td>Electron Microscopy Sciences</td><td>26385-02</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Orcein, 0.2%</td><td>Electron Microscopy Sciences</td><td>26385-03</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Hematoxylin alcoholic, 5%</td><td>Electron Microscopy Sciences</td><td>26385-04</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Ferric chloride, 10%</td><td>Electron Microscopy Sciences</td><td>26385-05</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Lugol&#039;s Iodine</td><td>Electron Microscopy Sciences</td><td>26385-06</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Woodstain scarlet-acid fuchsin working solution</td><td>Electron Microscopy Sciences</td><td>26385-07</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Acetic acid, 0.5%</td><td>Electron Microscopy Sciences</td><td>Various</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Phosphotungstic acid, 5%</td><td>Electron Microscopy Sciences</td><td>26385-09</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcoholic saffron, 6%</td><td>Electron Microscopy Sciences</td><td>26385-10</td><td>For Movat&#039;s Pentachrome staining</td></tr></tbody></table>

murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

Nach Drahtverletzung entwickelt Neointimahyperplasie im Laufe der Zeit und wird in der Regel nach 14 bis 28 Tagen untersucht. Die in dieser Arbeit führen zu robusten Generation von Intimahyperplasie bei Mäusen, wie dargestellt die histologischen Ergebnisse in Abbildung 3 beschriebenen Techniken. Ein unverletzt Femoralarterie intakt elastischen Lamellen und eine normale Dicke und Umfang demonstrieren. Ein verletzter Femoralarterie wird Intimahyperplasie zeigen, abgebaut elastischen Lamellen und zeigen ...

Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Mausmodell der Femoralarterie Draht Verletzung mit Implantation einer Perivaskuläre Drug Delivery-Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

murine-model-femoral-artery-wire-injury-with-implantation

Research

JoVE Journal

Medicine

17.9K Aufrufe.  University of Texas at Austin. We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis. 

Serie: Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Programmierung Stammzellen für therapeutische Angiogenese mit biologisch abbaubaren Polymernanopartikel

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Forschung

Biotechnik

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Osmotic Drug Delivery zu ischämischen Hinterbeine und Perfusion von Blutgefäßen mit Microfil für Mikro-Computertomographie

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Medizin

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

Ein Radio-Telemetrie-System zur Herz-Kreislauf-Funktion bei Ratten mit Rückenmarksdurchtrennung und embryonalen Neural Stem Cell Grafts überwachen

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Ein Mausmodell von arterieller Restenose: Technische Aspekte der Femoral Draht Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Ein Hydrogel Konstruieren und Fibrin-basierte Kleber Umgangs Therapeutics in einem murinen Myokardinfarkt Modell liefern.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induktion von beschleunigter Atherosklerose bei Mäusen: Das "Wire-Injury"-Modell

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Patch-Angioplastie in der Ratte Aorta oder Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplantation eines biobedruckten 3D-Patches in einem Murine-Modell des Myokardinfarkts

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

Implantation eines Tissue-Engineering-Gefäßtransplantats in die Halsschlagader der Maus mittels Manschettentechnik

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Multiphotonen-Intravital-Bildgebung zur Überwachung der Leukozytenrekrutierung während der Arteriogenese in einem murinen Hintergliedmaßenmodell

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...