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.

NOTA: Tutti i metodi indicati in questo protocollo sono stati approvati dalla cura degli animali e uso Comitato Istituzionale. 1. Preparazione del tavolo operatorio <ol><li> Impostare il pad di riscaldamento con ricircolo acqua calda sul tavolo operatorio, al microscopio dissezione. Posizionare la piastra di base in acciaio inox sul pad di riscaldamento. Posizionare un tampone assorbente sterile sulla piastra di base e piastra elettrica. </li><li> Disporre di strumenti chirurgici sterili su un tampone assorbente sterile adiacente. Raccogliere due coppie di angolari pinza a punta fine, due paia di pinze ad angolo, tre pinze emostatiche, due fili divaricatori, piccole forbici chirurgiche, micro-forbici, una angioplastica filo (diametro 0,15 pollici) e tre segmenti da 6 pollici di sutura 6.0 seta . </li><li> Piegare l&#39;estremità rotonda del filo angioplastica per abbinare la curvatura dell&#39;arteria femorale. In questo modo sarà più facile per far avanzare il filo durante l&#39;intervento chirurgico. </li></ol> 2. Preparazione diMouse for Surgery <ol><li> Anestetizzare un mouse per inalazione continua di 2,5% isoflurano. Assicuratevi di monitorare lo stato dell&#39;animale durante tutta la procedura. Eseguire un test pizzico di piede del mouse per confermare che è completamente anestetizzato. Assicurarsi che l&#39;animale non si muove quando il test pinch viene somministrato. </li><li> Applicare una pomata lubrificante per gli occhi dell&#39;animale per prevenire l&#39;essiccamento. Fissare l&#39;animale in posizione supina al tampone assorbente con del nastro chirurgico. Continuare la somministrazione 2,5% isoflurano via cono. </li><li> Usando una crema depilatoria, rimuovere la pelliccia dalla gamba e addome per la linea mediana. Sciacquare bene la pelle con acqua. Depilazione può essere eseguito un giorno prima dell&#39;intervento. Si noti che il trattamento eccessivo con la crema può provocare irritazione della pelle. </li><li> Immediatamente prima dell&#39;intervento chirurgico, applicare iodopovidone alla zona depilata con un applicatore con punta di cotone per sterilizzare la pelle. Sciacquare la pelle depilata con il 70% di etanolo e seccocon un applicatore con punta di cotone sterile. Ripetere 3 volte. </li><li> Somministrare una dose pre-operatoria di 5 mg / kg di carprofen tramite iniezione sottocutanea. </li></ol> 3. Isolamento dell&#39;arteria femorale <ol><li> Utilizzando piccole forbici chirurgiche, fare una incisione curva in pelle sopra l&#39;arteria femorale. Senza mezzi termini sezionare e fissare tessuti circostanti con divaricatori fissatori riavvolgitore magnetica per individuare l&#39;arteria femorale. Inumidire i tessuti periodicamente con soluzione salina per l&#39;irrigazione Applicare salina con un cotton fioc sterili. </li><li> Isolare l&#39;arteria femorale con pinze. Separare delicatamente il nervo dal fascio vascolare con pinze dalla punta sottile. Evitare di pungere la vena, e non danneggiare il nervo. Spingere il nervo lontano dal fascio per evitare di stimolare esso. </li><li> Separare delicatamente la vena femorale dalla arteria femorale, localizzare la biforcazione femorale. La regione della biforcazione è particolarmente difficile da sezionare. </li><li> Posterior alla biforcazione, ciclo di sutura 6.0 seta sotto l&#39;arteria femorale e sicuro con una pinza emostatica. Questo sutura prossimale verrà utilizzato per limitare il flusso sanguigno nell&#39;arteria. NOTA: C&#39;è una leggera variazione dei legami quando si esegue il metodo di legatura rispetto al metodo di cauterizzazione (vedi Figura 1 e Figura 2). </li><li> Distale alla biforcazione, ciclo 6.0 sutura di seta sotto l&#39;arteria femorale e sicuro con una pinza emostatica. Questo distali aiuti sutura nel posizionamento dell&#39;arteria. </li><li> Loop due punti di sutura sotto il ramo muscolare dell&#39;arteria femorale, li pre-cravatta e sicuro con una pinza emostatica. Ricordarsi di bagnare i tessuti con soluzione fisiologica. Se si esegue il metodo cauterizzazione di lesioni filo, solo un sutura in loop è necessario sul ramo muscolare. </li></ol> 4. Prestazioni di dall&#39;arteria femorale Wire Injury <ol><li> Limitare il flusso sanguigno nell&#39;arteria femorale tirando la sutura prossimale. Leggermente tirare il hemost distalein e hemostat garantire il ramo per esporre il sito per il arteriotomia. Legare il ramo muscolare legando la sutura intorno ad esso. NOTA: Sollevamento l&#39;arteria verso l&#39;alto in modo più efficace limitare il flusso di sangue che tirare da solo in senso orizzontale. </li><li> Sever il piccolo ramo con la cauterizzazione tra i due punti di sutura. Utilizzando micro-forbici, eseguire un arteriotomia nel ramo laterale della biforcazione. Utilizzare pinze in equilibrio su un rotolo di nastro adesivo chirurgico per stabilizzare le micro-forbici. </li><li> Confermare la presenza della arteriotomia usando pinze dalla punta sottile. Sollevare delicatamente l&#39;apertura del arteriotomia con le pinze. Introdurre l&#39;estremità arrotondata del filo nel arteriotomia con pinze. Per facilitare l&#39;avanzamento del filo, aggiungere uno o due gocce di lidocaina alla regione con una siringa. </li><li> Quando il filo raggiunge la sutura prossimale, rilasciare la sutura e regolare in modo che esso non può impedire l&#39;avanzamento del filo. Inserire il filo fino a quando non può avanzare ulteriormente. La punta di the filo dovrebbe fermarsi nella regione del legamento inguinale. </li><li> Lasciare che il filo di rimanere in arteria femorale per un minuto. Dopo un minuto, retrarre e far avanzare il filo in un movimento di taglio dieci volte di ferire e spogliare l&#39;endotelio dell&#39;arteria femorale. Per una lesione di minore gravità, diminuire il numero di volte che il filo è tirato dentro e fuori l&#39;arteria o lasciare il filo nell&#39;arteria solo per 1 min. </li><li> Ritrarre il filo lentamente. Quando l&#39;estremità arrotondata del filo ha superato la sutura prossimale, limitare il flusso nell&#39;arteria tirando la sutura prossimale. Rientrare completamente il filo. </li></ol> 5. legatura Branch Muscular <ol><li> Stringere la sutura rimanente sul ramo muscolare. Questo consentirà di evitare il sanguinamento dal arteriotomia. Flusso di ritorno al ramo muscolare, e confermare che il sangue non perde da esso. Tagliare le estremità delle suture sul ramo muscolare. </li></ol> 6. Metodo alternativo: localizzato Cauzazione del arteriotomia NOTA: Un approccio alternativo può essere presa per evitare la legatura del ramo muscolare e consentire l&#39;accesso vascolare attraverso il principale grande arteria femorale. <ol><li> Partendo da 4.1, per limitare il flusso femorale tirando sutura prossimale. Estrarre la sutura distale e la sutura fissaggio leggermente ramo muscolare per esporre il sito per l&#39;arteriotomia (Figura 1 e Figura 2). </li><li> Utilizzando micro-forbici, eseguire un arteriotomia al punto di diramazione dell&#39;arteria femorale e il ramo muscolare. Se l&#39;incisione viene effettuata nel lato dell&#39;arteria, può essere più facile per cauterizzare. Utilizzare pinze in equilibrio su un rotolo di nastro adesivo chirurgico per stabilizzare le micro-forbici. </li><li> Per verificare il successo di cauterizzazione, ripristinare il flusso per l&#39;arteria femorale allentando la sutura prossimale. Se l&#39;emorragia si verifica dal arteriotomia, ripetere la cauterizzazione. Se la cauterizzazione ha successo, il flusso di sangue sarà rimemorizzata distale al arteriotomia. </li><li> Introdurre il filo ed eseguire il pregiudizio filo come descritto nella sezione 4. </li><li> Continuare limitando il flusso di sangue nell&#39;arteria femorale. Scaldare una cauterizzazione a punta fine almeno 6 centimetri di distanza dal mouse. Come la punta cauterizzazione raffredda, applicare leggermente i lati della arteriotomia per chiudere l&#39;incisione. </li><li> Per verificare il successo di cauterizzazione, ripristinare il flusso per l&#39;arteria femorale allentando la sutura prossimale. Se l&#39;emorragia si verifica dal arteriotomia, ripetere la cauterizzazione. Se la cauterizzazione è successo, il flusso di sangue verrà ripristinato distale al arteriotomia. Rimuovere il prossimale temporanea e legami distali. </li></ol> 7. L&#39;impianto di Perivascolare Drug Delivery Patch <ol><li> Creare un cerotto farmaco perivascolare può essere creato come descritto in precedenti studi 11 o utilizzando metodi simili. Blunt angoli della regione di barriera della patch drug delivery usando forbici sterili </li><li> Posizionare il pat consegna della drogach sopra l&#39;arteria femorale feriti con il lato di rilascio farmaco rivolto l&#39;arteria. Se necessario, utilizzare pinze per migliorare la posizione della patch. </li></ol> 8. ferita Chiusura e recupero <ol><li> Chiudere la ferita con una semplice sutura interrotta composto da nodi quadrati. Una sutura discontinua prolunga chiusura della ferita in caso l&#39;animale cerca di rimuovere la sutura. </li><li> Spegnere l&#39;anestesia e rimuovere l&#39;animale dal set up. Lasciare che l&#39;animale di recuperare sul pad riscaldamento. </li><li> Continuare a monitorare il recupero del mouse. Controllare il sito di incisione ogni giorno per garantire che rimanga chiuso. Per il trattamento del dolore post-operatorio, somministrare una iniezione sottocutanea di 5 mg / kg di carprofen ogni 12 ore dopo l&#39;intervento chirurgico e poi ogni 12 ore per 2 giorni. Se il dolore continua oltre i primi 2 giorni, consultare un veterinario per le indicazioni su ulteriori antidolorifici. </li></ol> 9. raccolta femorale arterie di Istologia <ol><li> A 28 giorni post-operatorio, effettuare anidride carbonica l&#39;eutanasia sul mouse. NOTA: La portata appropriata di anidride carbonica dovrebbe sostituire 10-30% della camera per minuto e varierà a seconda delle dimensioni della camera utilizzata. Taglio del diaframma e puntura cardiaca dovrebbe essere eseguita come metodo secondario dell&#39;eutanasia. </li><li> Fissare il mouse in posizione supina con del nastro chirurgico. Praticare un&#39;incisione sopra l&#39;arteria femorale, dove è stata fatta l&#39;incisione chirurgica iniziale. </li><li> Per entrambi l&#39;arteria feriti e l&#39;arteria illeso dalla gamba controlaterale, senza mezzi termini sezionare e fissare i tessuti con divaricatori e fissatori riavvolgitore magnetici per individuare l&#39;arteria femorale circostante. Inumidire i tessuti periodicamente con soluzione salina per l&#39;irrigazione. Applicare soluzione salina con un cotton fioc sterili. Fare attenzione a non danneggiare l&#39;arteria. </li><li> Dopo l&#39;arteria femorale è stato isolato dal sito di arteriotomia originale dell&#39;aorta addominale, legare un segzione di sutura di seta nei pressi del sito originale del arteriotomia. Questo sutura identificare la fine più distale dell&#39;arteria femorale e facilitare la manipolazione del campione. </li><li> Utilizzando le forbici micro-dissezione di asportare l&#39;arteria femorale. Effettuare una incisione distale alla sutura. Effettuare l&#39;altro un&#39;incisione sull&#39;estremità opposta dell&#39;arteria femorale, accanto dell&#39;aorta addominale. </li><li> Trasferire l&#39;arteria asportato ad una piastra di Petri di vetro contenente soluzione fisiologica. Sezionare l&#39;arteria più per rimuovere il tessuto connettivo in eccesso o di grasso. Rimuovere delicatamente il sangue dal lume risciacquando con soluzione salina. </li><li> Trasferire l&#39;arteria di un flaconcino da 10% formalina tamponata. Conservare il flacone a 4 ° C con una leggera oscillazione per 48 ore. </li><li> Trasferire l&#39;arteria fissato al 70% di etanolo, per memorizzare fino alla trasformazione per l&#39;istologia. </li><li> Incorpora l&#39;arteria in blocchi di paraffina e la sezione dei blocchi per la colorazione. </li><li> Eseguire colorazione istochimica e immunochimico per valutare l&#39;entità del danno e intimale hyperplasia. NOTA: Per i risultati rappresentativi, abbiamo usato ematossilina e eosina di visualizzare i nuclei e la morfologia generale o macchia Pentachrome di un Movat per visualizzare le lamelle elastiche e altri componenti arteriose. </li></ol>

<ol>
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R., 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=Estrogen+inhibits+the+response-to-injury+in+a+mouse+carotid+artery+model.">Estrogen inhibits the response-to-injury in a mouse carotid artery model.</a> J Clin Invest. 96 (5), 2482-2488 (1995).</li><li>Yin, Y., Zhao, X., Fang, Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+artery+wire+injury+mouse+model+with+a+nonmicrosurgical+procedure.">Carotid artery wire injury mouse model with a nonmicrosurgical procedure.</a> Vascular. 18 (4), 221-226 (2010).</li><li>Nam, D., 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=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li><li>Nam, D., 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+model+of+disturbed+flow-induced+atherosclerosis+in+mouse+carotid+artery+by+partial+ligation+and+a+simple+method+of+RNA+isolation+from+carotid+endothelium.">A model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of RNA isolation from carotid endothelium.</a> J Vis Exp. 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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 border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:239px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Straight spring wire, 0.15&#8221; diameter</td>
			<td style="width:239px;">Cook</td>
			<td style="width:239px;">G02426</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">High Temperature cautery</td>
			<td style="width:239px;">Bovie Medical Corp.</td>
			<td style="width:239px;">HIT1</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">High-temperature fine tip for cautery</td>
			<td style="width:239px;">Bovie Medical Corp.</td>
			<td style="width:239px;">H101</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Micro-scissors</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">15000-13</td>
			<td style="width:239px;">For performance of arteriotomy</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Angled fine-tipped forceps</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">11251-35</td>
			<td style="width:239px;">For blunt dissection of vascular bundle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Angled forceps</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-5069</td>
			<td style="width:239px;">For clearing tissues</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Surgical scissors</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-5840</td>
			<td style="width:239px;">For cutting skin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Retractor</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-10</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Retractor wire</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-05</td>
			<td style="width:239px;">Attached to retractor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Base plate</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-03</td>
			<td style="width:239px;">For use with retractor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Magnetic retractor fixator</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-01</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Needle Holder</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-7822</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Hemostatic forceps</td>
			<td style="width:239px;">Biomedical Research Instruments, Inc.</td>
			<td style="width:239px;">34-1000</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Dissecting microscope</td>
			<td style="width:239px;">Meiji Techno</td>
			<td style="width:239px;">EMZ-5TR</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Microscope light source</td>
			<td style="width:239px;">Meiji Techno</td>
			<td style="width:239px;">FT191</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Warm water recirculator</td>
			<td style="width:239px;">Gaymar</td>
			<td style="width:239px;">TP-500</td>
			<td style="width:239px;">For maintaining mouse body temperature</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Reusable heating pad</td>
			<td style="width:239px;">Gaymar</td>
			<td style="width:239px;">TP-R 22G</td>
			<td style="width:239px;">For maintaining mouse body temperature</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Lidocaine</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">4.0 Vicryl suture with half circle needle</td>
			<td style="width:239px;">Ethicon</td>
			<td style="width:239px;">J494G</td>
			<td style="width:239px;">For post-surgical wound closure</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Sterile cotton-tipped applicators</td>
			<td style="width:239px;">Puritan</td>
			<td style="width:239px;">25-806-2WC</td>
			<td style="width:239px;">For application of depilatory cream and absorbing fluids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Depilatory cream</td>
			<td style="width:239px;">Nair</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Isoflurane</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Betadine</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">70% ethanol</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">6.0 braided silk suture</td>
			<td style="width:239px;">Teleflex Medical</td>
			<td style="width:239px;">4-S</td>
			<td style="width:239px;">For isolation of femoral artery during surgery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">0.9% sodium chloride</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;">For irrigating tissues</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Gel eye lubricant</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Glass petri dish</td>
			<td style="width:239px;">Pyrex</td>
			<td>3160-60</td>
			<td>For femoral artery harvest</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">10% buffered formalin</td>
			<td style="width:239px;">Various</td>
			<td></td>
			<td style="width:239px;">For fixation of femoral artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">70% ethanol</td>
			<td style="width:239px;">Various</td>
			<td></td>
			<td style="width:239px;">For fixation of femoral artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Bouin&#39;s fluid</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td></td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alcian blue, 1%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-01</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alkaline alcohol</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-02</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Orcein, 0.2%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-03</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Hematoxylin alcoholic, 5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-04</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Ferric chloride, 10%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-05</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Lugol&#39;s Iodine</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-06</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Woodstain scarlet-acid fuchsin working solution</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-07</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Acetic acid, 0.5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>Various</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Phosphotungstic acid, 5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-09</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alcoholic saffron, 6%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-10</td>
			<td style="width:239px;">For Movat&#39;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.

A seguito di infortunio filo, iperplasia neointimale si sviluppa nel tempo e in genere viene esaminata dopo 14 a 28 giorni. Le tecniche descritte in questo cavo lavoro robusta generazione di iperplasia intimale nei topi come mostrato risultati istologici della Figura 3. Un femorale indenne dimostreranno intatta lamelle elastiche e spessore normale e la circonferenza. Una arteria femorale infortunato mostrerà iperplasia intimale, degradato lamelle elastiche e dimostrare nuovamente endotelizzazione in mo...

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.

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. ...

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

Research

JoVE Journal

Medicine

17.9K Views.  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. 

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

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Contrazione Arteria mesenterica e Studi Relax Utilizzando Myography automatica Wire

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Ricerca

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 ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Un modello murino di ischemia miocardica e riperfusione lesioni da legatura della arteria discendente anteriore sinistra

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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.

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Modello murino di ischemia-riperfusione intestinale Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

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. ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Trombosi dell'arteria carotica canini cloruro ferrico-indotta: Un grande modello animale di danno vascolare

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Una lesione segmentale del palloncino segmentale controllato dalla pressione carotide del ratto con applicazione terapeutica periadventitiale

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Consegna intramiocardica ritardata di cellule staminali dopo l'infortunio di ricottamento ischemia in un modello Murine

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...