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.

NOTA: Etica Dichiarazione: Tutte le procedure sono conformi alla Guida per la cura e l&#39;uso di animali da laboratorio (National Academies Press, 8 ° edizione, 2011), e dei protocolli approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale presso l&#39;Università del Wisconsin. Tutti gli interventi chirurgici sono stati eseguiti in anestesia isoflurano (attraverso inalazione, portata 2 ml / min), e sono stati fatti tutti gli sforzi per ridurre al minimo le sofferenze. Gli animali sono stati sacrificati in una camera progressivamente riempita con CO 2. 1. L&#39;induzione di anestesia generale e incisione cutanea <ol><li> Utilizzare maschio C57BL / 6 topi con un&#39;età di 12-16 settimane e peso di 23-28 g. Anestetizzare l&#39;animale da isoflurano attraverso un apparato anestesia a circuito chiuso. Verificare il corretto anestesia pizzicando la sua punta. Applicare un po &#39;lacrime artificiali pomata sugli occhi per prevenire la secchezza. </li><li> Posizionare il mouse in posizione supina con dell&#39;arto posteriore sinistro leggermente addotto e il ginocchio sl congiuntaightly flesso. Radersi i capelli intorno alla regione coscia sinistra e disinfettare la pelle con clorexidina, applicato tre volte utilizzando cotone sterile applicatori con punta. Utilizzare una perlina sterilizzatrice calda per mantenere gli strumenti sterilizzati durante l&#39;intervento chirurgico. </li><li> Posizionare un drappo garza sterile sulla gamba sinistra. Effettuare una retta 1,5 centimetri, incisione cutanea longitudinale sulla coscia mediale sinistro dal ginocchio all&#39;inguine (Figura 1A). </li></ol> 2. L&#39;esposizione di dall&#39;arteria femorale e Filiali NOTA: La figura 1B mostra l&#39;anatomia vascolare lordo nella coscia superiore. L&#39;arteria femorale superficiale (SFA) può essere osservato attraverso un microscopio da dissezione quando l&#39;incisione cutanea è correttamente effettuata lungo la linea mediana della coscia mediale. <ol><li> Aprire la sottile fascia sovrastante la SFA con le pinzette sottili e poi avvolgere il SFA con 9-0 nylon sutura. NOTA: In questo processo, il nervo femorale è facilmente messo comeide, ma la vena femorale adiacente richiede molta attenzione perché la sua parete è così sottile che fora e sanguina facilmente. La tecnica chiave è quello di concentrarsi sulla arteria stessa; finché la dissezione avviene vicino all&#39;arteria, danni accidentali vena è evitabile. </li><li> Una volta che la SFA è in loop, tenere la sutura con una pinza di zanzare e tirarla leggermente verso il basso. Questa manovra rende ulteriormente dissezione più facile, ma per evitare di danneggiare l&#39;arteria non tirare troppo forza. </li><li> Continuare dissezione dell&#39;arteria prossimale per esporre e ciclo l&#39;arteria femorale comune (CFA) con 9-0 nylon sutura. NOTA: In genere, looping il CFA in un punto distale all&#39;arteria epigastrico (Figura 1B) è sufficiente per l&#39;inserimento del filo. Tenere il ciclo di sutura CFA con pinze zanzara nello stesso modo. </li><li> Tirare l&#39;anello di sutura CFA verso l&#39;alto e il ciclo di sutura SFA verso il basso per scoprire la biforcazione di SFA e profonda arteria femorale (DFA). Il DFAE, la principale filiale del TUF,va verso il muscolo della coscia (Figura 1B). NOTA: Il filo verrà inserito dal DFA tardi. </li><li> Loop la DFA con cautela perché la vena adiacente è aggrovigliato sotto l&#39;arteria. In caso di sanguinamento dalla vena, un 30- a 60 sec compressione semplice con una garza di solito è sufficiente a produrre l&#39;emostasi. </li><li> Una volta che il DFAE è in loop con 9-0 sutura in nylon, assicurarsi che tutti e tre i cicli di sutura sono al loro corrette posizioni (Figura 2A). </li><li> Procedere con la dissezione del DFA distalmente per legare l&#39;arteria in un punto più lontano possibile dalla biforcazione. Tagliare sutura in più, ma lasciare una certa lunghezza in modo che le due estremità possono essere tenuti con una pinza di zanzara, che stabilizzano il DFAE rende l&#39;inserimento filo più facile. </li></ol> 3. Creazione Wire Injury <ol><li> Inserire Wire via DFA <ol><li> Scolare le suture CFA, SFA, e DFA di stabilire un emostatico temporanea. </li><li> Fai un piccolo foro nel DFA con microscissors. Inserire le punte delle microtweezers curvi per dilatare il foro abbastanza grande per un filo di diametro 0,015 pollici (Figura 2B mostra la differenza di dimensioni tra l&#39;arteria e il filo). <ol><li> Prima di fare un buco nel DFA, allentare il cappio di sutura CFA e delicatamente bloccare / spremere il DFA più volte con microtweezers sordo-punta. Questa manovra si espande il diametro delle arterie notevolmente per facilitare l&#39;inserimento del filo. Inumidire accuratamente il filo con PBS è anche importante per un inserimento scorrevole. </li><li> Quando si effettua un foro nel DFA, non tagliare la parete arteriosa, ma fare un piccolo morso a forma di V e pizzicare la patta con microtweezers taglienti a punta per la manipolazione. </li></ol></li><li> Inserire il filo attraverso il foro adeguato, dilatato in DFA. Avanzare con delicatezza il filo verso l&#39;arteria iliaca mentre allentando l&#39;anello di sutura intorno al CFA. 5-8 mm è sufficiente a coprire tutta la lunghezza CFA in molti casi (Figura 2C </strong&gt;). Non inserire il filo con forza come una perforazione arteriosa solito si traduce in perdita del mouse. Accorciando il filo a circa 15 mm di lunghezza aiuta con movimentazione. </li></ol></li><li> Lasciare filo all&#39;interno per 1 min <ol><li> Dopo aver inserito il filo alla lunghezza ottimale, lasciare per 1 min per deformare eccessivamente la parete arteriosa. Inumidire il filo e le arterie con PBS per evitare l&#39;essiccazione. Un&#39;altra sutura 9-0 nylon può essere collocato intorno prossimale DFA al punto di inserimento filo durante questo tempo di attesa. Questo sutura verrà utilizzato successivamente per chiudere il foro sul DFA dopo la rimozione del filo. </li></ol></li><li> Recuperare Wire e riprendere il flusso di sangue <ol><li> Tenere il filo con una pinza nave incannulamento, e tirarlo indietro delicatamente; il filo deve essere recuperato senza problemi. Per superare alcuna resistenza, di solito causato dalla aderenza del filo alla parete arteriosa, tirando indietro il filo con rotazione dolce è utile. Ancora una volta, una manovra forzata potrebbe provocare la perdita animali. </li><li> Laopo il filo recuperato, legare il DFA nel punto di biforcazione con 9-0 sutura di nylon. Non legare troppo vicino alla biforcazione, che metterebbe a repentaglio il deflusso di sangue al SFA. </li><li> Allentare gli anelli di sutura SFA e CFA di riprendere il normale flusso sanguigno. Si noti che il CFA è ben espansa-by inserimento del filo (Figura 2D). </li></ol></li></ol> 4. Pelle Chiusura e postoperatoria cura <ol><li> Controllo sanguina. Stillicidio Minor può essere fermato con una semplice compressione; alcuni sanguinamenti maggiori arterie richiedono punti di sutura con una sutura 11-0 nylon. Queste arterie suturate finirà per determinare occlusione arteriosa. </li><li> Chiudere la pelle ferita con cucitrici di pelle (di solito 3-4 punti metallici). Mantenere l&#39;area ferita sterilizzata applicando soluzione chirurgica clorexidina. </li><li> Applicare trattamenti post-chirurgici. Controllare il dolore del mouse con iniezioni sottocutanee di buprenorfina. Tenere il mouse sul tappetino di riscaldamento fino a che non ha recuperato sufciente coscienza per mantenere decubito sternale. Casa in una gabbia pulita senza compagnia di altri animali fino pienamente recuperato. </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.

La procedura lesioni filo è applicabile a tutti i ceppi di topi, purché le strutture anatomiche sono simili. 4 In questo lavoro, abbiamo utilizzato maschio C57BL / 6 topi con un&#39;età di 12-16 settimane. Come abbiamo imparato dai nostri studi preliminari, le arterie femorali di topi più giovani di 10 settimane sono spesso così piccole che l&#39;inserimento del filo è molto impegnativo. D&#39;altra parte, l&#39;inserimento del filo in topi vecchi a 16 settimane è tecnicamente più semplice, ma tende a...

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

Le malattie cardiovascolari causate da aterosclerosi è la principale causa di morte nel mondo sviluppato. Restringimento del lume del vaso, a causa dello sviluppo della placca aterosclerotica o la rottura delle placche stabiliti, interrompe il normale flusso di sangue che porta a varie patologie, come l&#39;infarto del miocardio e ictus. Nella clinica procedure endovascolari, come l&#39;angioplastica sono comunemente eseguiti per riaprire il lume. Tuttavia, questi trattamenti inevitabilmente danneggiano la parete del serbatoio, nonché l&#39;endotelio vascolare, innescando una risposta guarigione eccessivo e lo sviluppo di una targa neointimale che si estende nel lume causando restenosi nave (ri-restringimento). Restenosi resta una delle principali cause di fallimento di trattamenti endovascolari per l&#39;aterosclerosi. Così, modelli animali preclinici di restenosi sono di vitale importanza per lo studio dei meccanismi fisiopatologici e traslazionale approcci per interventi vascolari. Tra diversi experime murinomodelli ntale, lesioni filo arteria femorale è ampiamente accettata come il più adatto per gli studi di restenosi post-angioplastica perché assomiglia da vicino la procedura di angioplastica che danneggia sia endotelio e parete del serbatoio. Tuttavia, molti ricercatori hanno difficoltà utilizzando questo modello grazie al suo elevato grado di difficoltà tecnica. Ciò è dovuto soprattutto un filo metallico deve essere inserito nell&#39;arteria femorale, che è circa tre volte più sottile il filo, per generare lesioni sufficiente ad indurre neointima prominente. Qui, descriviamo i dettagli chirurgici essenziali per superare efficacemente le maggiori difficoltà tecniche di questo modello. Seguendo le procedure presentate, eseguendo il pregiudizio del mouse filo femorale diventa più facile. Una volta familiarizzato, l&#39;intera procedura può essere completata entro 20 min.

Quattro settimane dopo l&#39;intervento, raccolgono l&#39;arteria femorale filo-feriti e rendono esemplare di blocco una paraffina per analizzare la formazione neointima. Indurre anestesia generale come descritto sopra. Aprire immediatamente il torace e inserire un calibro 20 farfalla ago al ventricolo sinistro. Collegare la linea ad una bottiglia di infusione. Eseguire la fissazione perfusione per infusione a goccia di PBS seguito da 4% paraformaldeide (di solito 10-20 ml, ciascuno). La bottiglia di infusione ...

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

Il mouse filo arteria femorale modello di lesione di restenosi è tecnicamente impegnativo. In questo protocollo vi mostriamo i principali dettagli tecnici essenziali per eseguire con successo danno filo da indurre neointima coerente per gli studi di restenosi.

Un modello murino di restenosi arteriosa: aspetti tecnici del femore Wire 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 Views.  University of Wisconsin School of Medicine and Public Health.  Il mouse filo arteria femorale modello di lesione di restenosi è tecnicamente impegnativo. In questo protocollo vi mostriamo i principali dettagli tecnici essenziali per eseguire con successo danno filo da indurre neointima coerente per gli studi di restenosi. 

Video: 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.

Modello murino di ischemia degli arti posteriori

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

Ricerca

Biologia

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.

Modello murino Spinotrapezius per valutare l&#39;impatto della legatura arteriolare sulla funzione microvascolare e rimodellamento

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

Medicina

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

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

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

Un modello murino di impianto di stent a livello dell&#39;arteria carotidea per lo Studio della restenosi

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.

Generazione e quantificazione 3-Dimensional di arteriosa lesioni nei topi mediante tomografia ottica di proiezione

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.

Modello murino di dall&#39;arteria femorale Wire Injury con impianto di una Perivascolare 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.

Induzione di aterosclerosi accelerata nei topi: il modello "Wire-Injury"

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.

Multiphoton Intravital Imaging per il monitoraggio del reclutamento dei leucociti durante l'arteriogenesi in un modello murino di arti posteriori

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.

Imaging tridimensionale ad alta risoluzione della vascolarizzazione del footpad in un modello di cancrena posteriore murina

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.

Lesione aortica di crusca a murina: un efficace<em&gt; In Vivo</em&gt; Modello di proliferazione cellulare liscia e funzione endoteliale

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.

Stabilire un modello di frattura del femore diafisario nei topi

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

Un modello murino di restenosi arteriosa: aspetti tecnici del femore Wire Injury

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Modello murino di dall'arteria femorale Wire Injury con impianto di una Perivascolare Drug Delivery Patch

Induzione di aterosclerosi accelerata nei topi: il modello "Wire-Injury"

Multiphoton Intravital Imaging per il monitoraggio del reclutamento dei leucociti durante l'arteriogenesi in un modello murino di arti posteriori

Imaging tridimensionale ad alta risoluzione della vascolarizzazione del footpad in un modello di cancrena posteriore murina

Lesione aortica di crusca a murina: un efficace

Stabilire un modello di frattura del femore diafisario nei topi