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

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

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>

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W., Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Rat+and+Mouse+Carotid+Artery+Surgery:+Injury+&amp;+Remodeling+Studies.">Experimental Rat and Mouse Carotid Artery Surgery: Injury &amp; Remodeling Studies.</a> ISRN Minim Invasive Surg. 2013, (2013).</li><li>Iafrati, M. 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=Estrogen+inhibits+the+vascular+injury+response+in+estrogen+receptor+alpha-deficient+mice.">Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.</a> Nat Med. 3 (5), 545-548 (1997).</li><li>Sullivan, T. 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. (40), (2010).</li><li>Kuhlmann, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+a+carotid+cuff+for+triggering+shear-stress+induced+atherosclerosis+in+mice.">Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.</a> J Vis Exp. (59), (2012).</li><li>Carmeliet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+wound+healing+and+neointima+formation+induced+by+perivascular+electric+injury+in+mice.">Vascular wound healing and neointima formation induced by perivascular electric injury in mice.</a> Am J Pathol. 150 (2), 761-776 (1997).</li></ol>

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

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

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

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

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

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

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

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

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

Cellule staminali per la programmazione terapeutica angiogenesi Uso biodegradabili polimerico Nanoparticelle

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

Ricerca

Bioingegneria

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

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

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

Osmotica Drug Delivery per arti posteriori ischemico e la perfusione dei vasi con Microfil per Micro-Tomografia Computerizzata Imaging

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

Medicina

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

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

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

Un sistema radio-telemetria per monitorare la funzionalità cardiovascolare in ratti con midollo spinale e Transection embrionali staminali neurali Innesti cellulari

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

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

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

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

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 ...

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

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

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

Un idrogel Costruire e fibrina-based Glue approccio per fornire Therapeutics in una murino Myocardial Infarction Model.

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

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. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

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

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

Patch Angioplastica nel ratto aorta o vena cava inferiore

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

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

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

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

Trapianto di una patch biostampa 3D in un modello Murine di infarto miocardico

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

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

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

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

Impianto di innesto vascolare di ingegneria tissutale nell'arteria carotide di topo tramite tecnica della cuffia

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

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

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

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

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 ...

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

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