Name: a murine model of arterial restenosis: technical aspects of femoral wire injury
Uploaded: 2015-03-10T15:00:00.000+00:00
Duration: 6 min 42 s
Description: Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

This work was supported by a Wisconsin Partnership Program New Investigator Award (ID 2832), a National Heart, Lung, Blood Institute R01 Grant (HL-068673) and a T32 training Grant (HL-110853). We thank Dr. Melina Kibbe&#8217;s group at Northwestern University for providing helpful information.

HINWEIS: Ethics Statement: Alle Verfahren entsprechen dem Leitfaden für die Pflege und Verwendung von Labortieren (National Academies Press, 8. Auflage, 2011), und Protokolle von der Institutional Animal Care und Verwenden Committee an der University of Wisconsin zugelassen. Alle Operationen wurden unter Isofluran-Narkose (durch Einatmen, Durchflussrate von 2 ml / min) durchgeführt, und alle Anstrengungen unternommen, um das Leiden zu minimieren. Die Tiere wurden in einer Kammer allmählich mit CO 2 gefüllt euthanasiert. 1. Narkoseeinleitung und Hautschnitt <ol><li> Verwenden männlichen C57BL / 6-Mäuse mit einem Alter von 12-16 Wochen und das Gewicht 23-28 g. Anesthetize das Tier durch Isofluran über einen geschlossenen Kreisnarkosegerät. Überprüfen Sie die Betäubung durch Kneifen seine Zehen. Tragen Sie etwas künstliche Tränen Salbe auf die Augen bis zur Trockenheit zu verhindern. </li><li> Platzieren Sie die Maus in Rückenlage mit dem linken Hinterbein leicht verschleppt und Kniegelenk slightly gebeugt. Rasieren Sie die Haare um den linken Oberschenkel Region und desinfizieren Sie die Haut mit Chlorhexidin, angewendet dreimal mit sterilen Wattestäbchen Applikatoren. Verwenden Sie eine heiße Perle Sterilisator, die Instrumente während der Operation sterilisiert zu halten. </li><li> Legen Sie eine sterilisierte Gaze Tuch über das linke Bein. Einen 1,5 cm gerader Längs Hautinzision links medialen Oberschenkel vom Kniegelenk auf der Leiste (1A). </li></ol> 2. Exposition der Femoralarterie und Zweige HINWEIS: 1B zeigt die Bruttogefäßanatomie in den Oberschenkel. Die oberflächliche Oberschenkelarterie (SFA) können durch ein Präpariermikroskop beobachtet, wenn die Inzision richtig entlang der Mittellinie des medialen Oberschenkel vorgenommen werden. <ol><li> Öffnen Sie die dünne Faszie über dem SFA mit feinen Pinzette und dann die Schleife die SFA mit 9-0 Nylonnaht. HINWEIS: Bei diesem Verfahren wird der N. femoralis ist einfach wie setzenide, aber die angrenzenden femoralis erfordert besondere Aufmerksamkeit, weil ihre Wand ist so dünn, dass es leicht perforiert und blutet. Der Schlüssel Technik ist es, auf die Arterie selbst zu konzentrieren; solange der Präparation wird in der Nähe der Arterie durchgeführt wird, ist jede versehentliche Venenschäden vermeidbar. </li><li> Sobald die SFA geschlungen, halten Sie die Naht mit Moskito Pinzette und ziehen Sie sie leicht nach unten. Dieses Manöver macht weitere Präparation leichter, aber um eine Beschädigung der Arterie nicht zu kräftig ziehen. </li><li> Weiter sezieren die Arterie proximal zu entlarven und Schleife die A. femoralis communis (CFA) mit 9-0 Nylonnaht. HINWEIS: Normalerweise Schleifen des CFA an einem Punkt distal der A. epigastrica (1B) ist ausreichend für Drahteinführung. Halten Sie die CFA Fadenschleife mit Moskito Pinzette auf die gleiche Weise. </li><li> Ziehen Sie den CFA Fadenschleife nach oben und der SFA Fadenschleife nach unten, um die Gabelung der SFA und tiefe Oberschenkelarterie (EDA) zu entdecken. Das EDA, der Hauptzweig der CFA,fließt in den Oberschenkelmuskel (1B). HINWEIS: Der Draht wird später von der DFA eingefügt werden. </li><li> Schleife das EDA mit besonderer Vorsicht, da die angrenzenden Vene unter der Arterie verheddert. Im Falle von Blutungen aus der Vene ist ein 30- bis 60 sec einfache Kompression mit Gaze Regel ausreichend, um die Hämostase zu erzeugen. </li><li> Sobald der DFA mit 9-0 Nylonnaht geschleift, sicherzustellen, dass alle drei Nahtschleifen sind an die richtige Position (Abbildung 2A). </li><li> Fahren Sie mit der Dissektion der DFA distal, um die Arterie an einer Stelle so weit wie möglich von der Gabelung zu ligieren. Geschnitten zusätzliche Naht lassen aber eine bestimmte Länge, so dass die beiden Enden mit Moskito Pinzette, die die Herstellung von DFA Drahteinsetzlöcher leichter Stabilisierung statt. </li></ol> 3. Erstellen von Draht Injury <ol><li> Legen Draht über DFA <ol><li> Belastung der CFA, SFA und DFA Nähten, um eine temporäre hemostat etablieren. </li><li> Machen Sie ein kleines Loch in der DFEin mit Mikroschere. Stecken Sie die Spitzen der gebogenen Mikropinzetten, um das Loch groß genug für einen Draht mit einem 0,015 Zoll Durchmesser erweitern (2B zeigt den Größenunterschied zwischen der Arterie und dem Draht). <ol><li> Bevor Sie ein Loch in der DFA, lösen Sie die CFA Fadenschleife und sanft klemmen / Drücken Sie die DFA mehrmals mit stumpfen bestückte Mikropinzetten. Dieses Manöver erweitert die arteriellen Durchmesser bemerkenswert, um die Drahteinführung zu erleichtern. Befeuchten Sie den Draht sorgfältig mit PBS ist auch wichtig für eine reibungslose Einsetzen. </li><li> Wenn ein Loch in der DFA, nicht beschneiden die Arterienwand, aber einen kleinen V-förmigen Spalt und nehmen Sie die Klappe mit spitzen Mikropinzetten für Manipulation. </li></ol></li><li> Führen Sie den Draht durch die angemessen-dilatative Loch auf der DFA. Den Draht vorsichtig voran in Richtung der Darmbeinarterie, während das Lösen der Fadenschleife um das CFA. 5-8 mm reicht aus, um alle CFA Länge in den meisten Fällen zu decken (2C </strong&gt;). Haben Sie das Kabel mit Gewalt einlegen nicht als ein arterieller Perforation führt in der Regel zum Verlust der Maus. Die Verkürzung der Draht bis ca. 15 mm in der Länge hilft bei der Handhabung. </li></ol></li><li> Lassen Draht innen für 1 Min <ol><li> Nach dem Einlegen der Kabel an die optimale Länge, lassen Sie es für 1 Minute, um die Arterienwand überfordern. Befeuchten Sie den Draht und Arterien mit PBS, um ein Austrocknen zu verhindern. Eine weitere 9-0 Nylonnaht können rund um die DFA proximal der Drahteinführungspunkt während dieser Wartezeit platziert werden. Dieser Faden wird später verwendet werden, um das Loch auf der DFA nach Entfernen des Drahtes zu schließen. </li></ol></li><li> Abrufen Draht und Fortsetzen der Durchblutung <ol><li> Halten Sie den Draht mit Schiff Kanülierung Pinzetten, und ziehen Sie sie vorsichtig zurück; der Draht sollte problemlos abgerufen werden. Keinen Widerstand, in der Regel durch die Einhaltung des Drahtes an der Arterienwand, zu überwinden, das Zurückziehen des Drahtes mit leichter Rotation hilfreich. Auch hier könnte eine kraftvolle Manöver in der Tierverlust führen. </li><li> Aach das Abrufen der Draht, ligieren die DFA am Verzweigungspunkt mit 9-0 Nylonnaht. Ligieren Sie nicht zu nahe an der Gabelung, die den Blutabfluss an die SFA in Frage stellen würde. </li><li> Lösen Sie die SFA und CFA Nahtschleifen, um den normalen Blutfluss wieder aufzunehmen. Beachten Sie, dass die CFA wird durch Drahteinführung (2D) gut ausgebaut. </li></ol></li></ol> 4. Hautverschluss und postoperative Pflege <ol><li> Steuer blutet. Minor Nässen durch einfache Kompression gestoppt werden; einige große arterielle Blutungen wird Stiche mit einem 11-0 Nylonfaden erfordern. Diese vernäht Arterien schließlich in Arterienverschluss führen. </li><li> Schließen Sie die Haut mit Hautklammergeräte (in der Regel 3-4 Heftklammern) gewickelt. Halten Sie die Wundfläche durch Anwendung Chlorhexidin chirurgische Lösung sterilisiert. </li><li> Bewerben postoperative Behandlungen. Steuern Sie den Schmerz der Maus mit subkutanen Injektionen von Buprenorphin. Halten Sie die Maus auf die Erwärmung pad bis sie suf wiederreichend Bewusstsein Brustlage zu halten. Haus in einem sauberen Käfig ohne Begleitung anderer Tiere, bis sie vollständig erholt. </li></ol>

<ol>
	<li>Dzau, V. J., Braun-Dullaeus, R. C., Sedding, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+proliferation+and+atherosclerosis:+new+perspectives+and+therapeutic+strategies.">Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.</a> Nat Med. 8 (11), 1249-1256 (2002).</li><li>Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+hyperplasia+in+murine+models.">Intimal hyperplasia in murine models.</a> Curr Drug Targets. 9 (3), 251-260 (2008).</li><li>Ferns, G. A., Avades, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanisms+of+coronary+restenosis:+insights+from+experimental+models.">The mechanisms of coronary restenosis: insights from experimental models.</a> Int J Exp Pathol. 81 (2), 63-88 (2000).</li><li>Sata, M., 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=A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia.">A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.</a> J Mol Cell Cardiol. 32 (11), 2097-2104 (2000).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Lv, L., Meng, Q., Ye, M., Wang, P., Xue, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT4+deficiency+protects+against+neointima+formation+following+arterial+injury+in+mice.">STAT4 deficiency protects against neointima formation following arterial injury in mice.</a> J Mol Cell Cardiol. 74C, 284-294 (2014).</li><li>Zhu, B., Kuhel, D. G., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E+inhibits+neointimal+hyperplasia+after+arterial+injury+in+mice.">Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.</a> Am J Pathol. 157 (6), 1839-1848 (2000).</li><li>Fu, Y., Duru, E. A., Davies, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+metabolic+syndrome+on+the+response+to+arterial+injury.">Effect of metabolic syndrome on the response to arterial injury.</a> J Surg Res. , (2014).</li><li>Kuhel, D. G., Zhu, B., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+in+genetic+determinants+for+injury-induced+neointimal+hyperplasia+and+diet-induced+atherosclerosis+in+inbred+mice.">Distinction in genetic determinants for injury-induced neointimal hyperplasia and diet-induced atherosclerosis in inbred mice.</a> Arterioscler Thromb Vasc Biol. 22 (6), 955-960 (2002).</li><li>Cooley, 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=Mouse+strain+differential+neointimal+response+in+vein+grafts+and+wire-injured+arteries.">Mouse strain differential neointimal response in vein grafts and wire-injured arteries.</a> Circ J. 71 (10), 1649-1652 (2007).</li><li>Ishigami, N., 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=Deficiency+of+CuZn+superoxide+dismutase+promotes+inflammation+and+alters+medial+structure+following+vascular+injury.">Deficiency of CuZn superoxide dismutase promotes inflammation and alters medial structure following vascular injury.</a> J Atheroscler Thromb. 18 (11), 1009-1017 (2011).</li></ol>

The authors declare no competing financial interests.

Der Draht Verletzungsverfahren ist für alle Stämme von Mäusen, solange ihre anatomischen Strukturen sind ähnlich. 4 In diesem Dokument verwendet man männliche C57BL / 6-Mäuse mit einem Alter von 12-16 Wochen. Wie wir von unseren Vorstudien gelernt, sind die Oberschenkelarterien von Mäusen, die jünger als 10 Wochen oft so klein, dass Drahteinführung ist eine ziemliche Herausforderung. Andererseits ist Drahteinsetzlöcher in Mäusen, die älter als 16 Wochen technisch einfacher, aber neigt dazu, in var...

In the era of expanded application of endovascular treatments for various cardiovascular diseases, restenosis after angioplasty is one of the major problems for patients undergoing such treatments. Damage to the vascular endothelium at the time of angioplasty, in concert with the atherosclerotic background, induces excessive smooth muscle cell proliferation in the medial layer, resulting in neointimal hyperplasia.1,2 A viable animal model that recapitulates post-angioplasty neointimal hyperplasia is, thus, important not only for the investigation of disease mechanisms but also for the development of effective therapeutics to treat this pathology.
Mice represent an excellent model animal to recapitulate neointimal hyperplasia for the following reasons: the genetic backgrounds of experimental mice are well established; a wide variety of genetically-modified strains are available;2 obtaining littermates of the same background is easy; and the cost of the animals is relatively low. Arterial ligation model and wire injury model are the two most common mouse models of mechanically-induced neointimal hyperplasia. The arterial ligation model is easy to create, but physiologically dissimilar to the actual angioplasty procedure. The wire injury model closely mimics actual angioplasty procedures but is technically difficult due to the small size of mouse arteries.2,3 Sata et al. first described a wire injury method for mouse femoral arteries based on the anatomical structure of the vasculature and the use of proper-sized flexible wire. Utilizing this technique, they succeeded in reproducibly inducing neointimal hyperplasia in various strains of mice.4
Although femoral wire injury is a well-established model, some of the technical aspects of the technique are highly challenging compared to other models such as ligation.5 The purpose of this paper is to describe our mouse wire injury model procedures in detail, which is a modified version of Sata&#8217;s original method. We have made two main modifications: 1) Looping only the arteries, and 2) No lidocaine use.

<table><tbody><tr><td>Fixed Core Wire Guide</td><td>Cook</td><td>G02426</td><td>Diameter 0.015 inch, Straight</td></tr><tr><td>Dilation Forceps</td><td>Roboz</td><td>RS-4927</td><td>Curved, blunt tips</td></tr><tr><td>Dumont Tweesers #5</td><td>World Precision Instruments</td><td>14095</td><td>Straight, sharp tips</td></tr><tr><td>Dumont Vessel Cannulation Forceps</td><td>World Precision Instruments</td><td>503373</td><td></td></tr><tr><td>McPherson-Vannas Scissors</td><td>World Precision Instruments</td><td>501234</td><td></td></tr><tr><td>Mosquito Forceps</td><td>World Precision Instruments</td><td>501291</td><td></td></tr><tr><td>Ethilon Nylon Suture 9-0</td><td>Ethicon</td><td>7717G</td><td>9-0, Black Nylon Monofilament</td></tr><tr><td>Micro AROSuture, Sterile 11-0, 70 micrometers, MET Point</td><td>AROSurgical</td><td>VT4A00N07</td><td>11-0, Black Nylon Monofilament</td></tr><tr><td>3M Precise Multi-Shot DS Disposable Skin Stapler</td><td>3M</td><td>DS-25</td><td></td></tr></tbody></table>

a murine model of arterial restenosis: technical aspects of femoral wire injury

Kardiovaskuläre Erkrankungen durch Arteriosklerose ist die häufigste Todesursache in der entwickelten Welt. Einengung des Gefäßlumens, aufgrund atherosklerotischen Plaque Entwicklung oder dem Aufbrechen der etablierten Plaques, unterbricht den normalen Blutfluss führt zu verschiedenen Begleiterkrankungen wie Herzinfarkt und Schlaganfall. In der Klinik endovaskulären Verfahren wie Angioplastie werden üblicherweise durchgeführt, um den Hohlraum wieder zu öffnen. Jedoch haben diese Behandlungen unvermeidbar beschädigt die Gefßwand sowie die Gefäßendothel Auslösen eines übermäßigen Heilungsreaktion und die Entwicklung einer neointimalen Plaque, die in das Lumen verursacht Gefäß Restenose (Wiederverengung) erstreckt. Restenose bleibt eine Hauptursache für Versagen endovaskulären Behandlungen für Atherosklerose. Somit sind vorklinischen Tiermodellen von Restenose für die Untersuchung der pathophysiologischen Mechanismen sowie Translationsansätze Gefäßinterventionen von entscheidender Bedeutung. Unter mehreren murinen Experimental Modellen wird Femoralarterie Drahtverletzung gilt als die am besten geeignete für die Untersuchung der Restenose nach Angioplastie akzeptiert, weil es ähnelt der Angioplastie-Verfahren, die sowohl Endothel und Gefäßwand verletzt. , Haben viele Forscher jedoch Schwierigkeiten Verwendung dieses Modell aufgrund seiner hohen technischen Schwierigkeitsgrad. Dies ist vor allem, weil ein Metalldraht muss in die Oberschenkelarterie, die etwa dreimal geringer als die Draht eingeführt werden, um eine ausreichende Verletzungen erzeugen prominente Neointima induzieren. Hier beschreiben wir die wesentlichen chirurgischen Details, die wichtigsten technischen Schwierigkeiten dieses Modells effektiv zu überwinden. Indem Sie die vorgestellten Verfahren, die Durchführung der Maus Femoralarterie Drahtverletzung wird einfacher. Sobald vertraut, kann das gesamte Verfahren innerhalb von 20 Minuten abgeschlossen werden.

Vier Wochen nach der Operation, zu ernten die Draht verletzten Oberschenkelarterie und machen einen Paraffinblock Probe, um die Neointimabildung analysieren. Induzieren Narkose wie oben beschrieben. Sofort Öffnen Sie die Kiste und legen Sie eine 20-Gauge-Butterfly-Nadel in die linke Herzkammer. Schließen Sie die Leitung an einer Infusionsflasche. Führen Perfusionsfixierung über Tropfinfusion von PBS, gefolgt von 4% Paraformaldehyd (in der Regel 10 bis 20 ml, jeweils). Die Infusionsflasche wird ca. 140 cm h?...

Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

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

Die Maus Femoralarterie Drahtverletzungsmodell der Restenose ist technisch anspruchsvoll. In diesem Protokoll zeigen wir die wichtigsten technischen Details unerlässlich für die erfolgreiche Durchführung von Draht Verletzung konsequente Neointima für Studien der Restenose zu induzieren.

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 ...

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Research

JoVE Journal

Medicine

20.1K Aufrufe.  University of Wisconsin School of Medicine and Public Health.  Die Maus Femoralarterie Drahtverletzungsmodell der Restenose ist technisch anspruchsvoll. In diesem Protokoll zeigen wir die wichtigsten technischen Details unerlässlich für die erfolgreiche Durchführung von Draht Verletzung konsequente Neointima für Studien der Restenose zu induzieren. 

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

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Mausmodell der hinteren Gliedmaßen Ischämie

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Forschung

Biologie

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

Murine Spinotrapezius Modell, um die Auswirkungen der Arteriolen Ligation auf mikrovaskuläre Funktion und Remodeling beurteilen

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

Medizin

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 ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generation und 3-Dimensional Quantifizierung der arterielle Läsionen in Mäuse mit optischen Projektionstomographie

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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.

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. ...

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. ...

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 ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Hochauflösende dreidimensionale Abbildung der Fußpolstervaskulatur in einem murinen Hinterbein-Gangränmodell

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Murine Aortic Crush Injury: Eine effiziente<em&gt; In vivo</em&gt; Modell der glatten Muskelzellproliferation und Endothelfunktion

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Etablierung eines diaphysären Femurfrakturmodells bei Mäusen

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...