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.

NOT: Bu protokolde gösterilen tüm yöntemler Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmıştır. Cerrahi Tablo 1. Hazırlık <ol><li> Diseksiyon mikroskobu altında, cerrahi masada sıcak su sirkülasyon ısıtma pedi ayarlayın. Isıtma pedi üzerine paslanmaz çelik taban plakası yerleştirin. Taban plakası ve ısıtma yastığı steril emici ped yerleştirin. </li><li> Bir komşu steril emici ped üzerine steril cerrahi aletler düzenleyin. Açılı ince uçlu forseps iki çift açılı forseps iki çift, üç hemostatik forseps, iki Retraktörler teller, küçük cerrahi makas, mikro-makas, bir anjiyoplasti tel (0.15 inç çap) ve 6.0 ipek sütür üç 6-inç segmentleri toplayın . </li><li> Femoral arter eğrilik maç anjiyoplasti tel yuvarlak ucunu bükün. Bu daha kolay ameliyat sırasında tel ilerletmek için yapacak. </li></ol> 2. HazırlıkCerrahi Fare <ol><li> % 2.5 izofluran sürekli inhalasyon yoluyla bir fare anestezisi. Prosedür boyunca hayvanın durumunu izlemek için emin olun. Tam anestezi olduğunu doğrulamak için fare ayak bir tutam testi yapın. Tutam testi uygulandığında hareket etmiyor hayvan sağlayın. </li><li> Kurumayı önlemek için hayvanın gözlerine bir yağlama merhem sürün. Cerrahi bant kullanarak emici ped ile yatar pozisyonda hayvan sabitleyin. Burun konisi üzerinden% 2.5 izofluran uygulanmasını devam edin. </li><li> Bir tüy dökücü krem ​​kullanmak, orta hattına bacak ve karın kürk çıkarın. Su ile iyice durulayın cildi. Depilasyon cerrahi bir gün önceden gerçekleştirilebilir. Cilt tahrişe yol açabilir krem ​​ile bu aşırı tedavi unutmayın. </li><li> Hemen ameliyat öncesi, cildi sterilize etmek için bir pamuk uçlu bir aplikatör ile tüy dökücü krem ​​alana povidon-iyot uygulayın. % 70 etanol ve kurutulur cımbız çiftleri cilt durulayınsteril pamuk uçlu bir aplikatör ile. 3 kez tekrarlayın. </li><li> Deri altına enjeksiyon ile karprofen 5 mg / kg&#39;lık bir ameliyat öncesi dozu uygulayın. </li></ol> Femoral Arter 3. İzolasyonu <ol><li> Küçük cerrahi makas kullanarak, femoral arter üzerinde deride kavisli bir kesi yapmak. Açık açık femoral artere bulmak için retraktörler manyetik retraktör fiksatörler kullanarak çevredeki dokuları incelemek ve güvenli. Steril pamuk uçlu bir aplikatör kullanılarak tuzlu uygulayın sulama için periyodik serum fizyolojik ile dokuları nemlendirin. </li><li> Forseps kullanarak femoral arter izole edin. Yavaşça ince uçlu forseps kullanılarak damar paket gelen sinir ayrı. Ven delinmesiyle kaçının ve sinir zarar vermez. Bunu önlemek için uyarıcı uzakta paket gelen sinir itin. </li><li> Yavaşça femur çatallanma yerini, femoral arter femoral ven ayrı. çatallanma bölgesi incelemek için özellikle zordur. </li><li> Posterior çatallanma, döngü hemostatla femoral arter ve güvenli altında 6.0 ipek sütür. Bu proksimal sütür arter kan akışını kısıtlamak için kullanılır. NOT: dağlama yöntemiyle karşı ligasyonu yöntemi yaparken bağları hafif bir varyasyon vardır (Şekil 1 ve Şekil 2). </li><li> Bifürkasyonun distalinde, döngü 6.0 ipek femoral arter altında dikiş ve hemostatla güvenli. Arter konumlandırma Bu uzak sütür yardımcıları. </li><li> Femoral arter kas dalı altında Döngü iki dikiş, onları ön-kravat ve bir hemostatla sabitleyin. Tuzlu su ile dokuları nemlendirmek için unutmayın. Tel yaralanma koter yöntemi gerçekleştirmek ise, yalnızca bir ilmekli dikiş kas şube gereklidir. </li></ol> Femoral Arter Tel Yaralanma 4. Performans <ol><li> Proksimal sütür çekerek femoral artere kan akışını kısıtlamak. Biraz uzak Memost çekinhemostat şube güvence altına ve arteriotomi için site ortaya çıkarmak. Etrafında dikiş bağlayarak kas şube Arter. NOT: yukarı arteri Kaldırma daha etkili yatay yalnız çekerek daha kan akışını kısıtlamak olur. </li><li> İki sütür arasındaki koter ile küçük şube Sever. Mikro-makas kullanarak, çatallanma yan dalında bir arteriyotomi gerçekleştirin. Mikro-makas stabilize etmek için cerrahi bant rulo üzerinde dengeli forseps kullanın. </li><li> Ince uçlu forseps kullanarak arteriotomi varlığını onaylayın. Yavaşça forseps Arteriyotomi açılmasını kaldırın. Forseps kullanarak arteriotomi içine tel yuvarlak ucunu tanıtın. Telin ilerlemesini kolaylaştırmak için, bir şırınga kullanılarak bölgeye lidokain, bir veya iki damla ekleyin. </li><li> Tel proksimal sütür ulaştığında, dikiş bırakın ve tel ilerleme engel olamaz ki ayarlayın. Daha da ileri edemez kadar tel yerleştirin. th ucue tel inguinal ligament bölgede durması gerekir. </li><li> Tel, bir dakika için femoral arter içine kalmasına izin verir. Zarar ve femoral arter endoteli denude için on kez bir dakika sonra, geri ve bir testere hareket tel ilerlemek. Daha az şiddette bir yaralanma için, tel ve arterin dışarı çekilir sayısını azaltmak veya sadece 1 dakika arterde tel bırakın. </li><li> Yavaş yavaş teli geri çekin. Tel yuvarlak ucu proksimal sütür geçtiğinde, proksimal sütür çekerek artere akışını kısıtlamak. Tamamen teli geri çekin. </li></ol> 5. Musküler Şubesi bağlanması <ol><li> Kas şube kalan dikiş sıkın. Bu arteriotomi kanamayı önlemek olacaktır. Kas şube akışını dönün ve bu kan ondan sızıntı değil onaylayın. Kas dalı üzerinde dikişler gelen uçlarını kesin. </li></ol> 6. Alternatif Yöntem: Lokalize Cauarteriyotomi yon NOT: Alternatif bir yaklaşım kas şube bağlanmasını önlemek ve daha büyük ana femoral arter yoluyla damar erişime izin vermek için alınabilir. <ol><li> 4.1 itibaren, proksimal sütür çekerek femoral artere akışını kısıtlamak. Distal sütür ve arteriotomi için site (Şekil 1 ve Şekil 2) ortaya çıkarmak için hafifçe kas şube güvence dikiş çekin. </li><li> Mikro-makas kullanarak, femoral arter ve kas şube şube noktada bir arteriyotomi gerçekleştirin. Kesi arter tarafında yapılır, bu dağlamak daha kolay olabilir. Mikro-makas stabilize etmek için cerrahi bant rulo üzerinde dengeli forseps kullanın. </li><li> Dağlama başarısını test etmek için, proksimal sütür gevşeterek femoral artere akışını geri. Kanama arteriotomi gelen oluşursa, koter tekrarlayın. Koter başarılı olursa, kan akışı yeniden edilecekarteriotomi distalinde saklı. </li><li> Tel tanıtılması ve bölüm 4&#39;te tarif edildiği gibi tel yaralanma yerine getirir. </li><li> Femoral artere kan akışını kısıtlayan devam edin. En az 6 inç uzakta fare ince uçlu koter ısıtın. Koter ucu soğudukça, kesi kapatmak için arteriotomi yan hafifçe uygulayın. </li><li> Dağlama başarısını test etmek için, proksimal sütür gevşeterek femoral artere akışını geri. Kanama arteriotomi gelen oluşursa, koter tekrarlayın. Koter başarılı olursa, kan akışı arteriotomi distalinde restore edilecek. Geçici yakın ve uzak bağları çıkarın. </li></ol> Perivasküler İlaç Dağıtım Patch 7. implantasyonu <ol><li> Daha önceki çalışmalarda 11 veya benzer yöntemler kullanılarak tarif edildiği gibi bir perivasküler ilaç yama oluşturma oluşturulabilir. Steril makaslar kullanılarak İlaç sağlama yaması hücresinden köşelerini kör </li><li> Ilaç dağıtım pat yerleştirinarter bakan ilaç salan tarafı yaralı femoral arter üzerine ch. Gerekirse, yama konumunu iyileştirmek için forseps kullanabilir. </li></ol> 8. Yara Kapatma ve Kurtarma <ol><li> Kare knot oluşan basit bir kesintiye dikiş ile yarayı kapatın. Bir süreksiz sütür hayvan dikiş kaldırmak için çalışır durumda yara kapanmasını uzatır. </li><li> Anestezi kapatın ve set up hayvan çıkarın. Hayvan ısınma pad üzerinde kurtarmak için izin verin. </li><li> Farenin kurtarma izlemeye devam edin. O kapalı kalmasını sağlamak için her gün kesi sitesini kontrol edin. Ameliyat sonrası ağrının tedavi edilmesi için, 2 gün sonra, her 12 saat cerrahi sonrası 5 mg / karprofen, her 12 saat kg deri altı enjeksiyon vermek ve. Ağrı ilk 2 gün ötesine devam ederse, daha fazla ağrı ilaç tarifini bir veteriner danışın. </li></ol> Histoloji 9. Hasat Femoral Arter <ol><li> Ameliyat sonrası 28 gün sonra, fare karbondioksit ötenazi yapmak. NOT: karbon dioksit uygun akış hızı, dakikada odasının% 10-30 yerinden olmalı ve kullanılan oda büyüklüğüne bağlı olarak değişecektir. Diyafram ve kalp ponksiyonu kesme ötenazi ikincil yöntem olarak yapılmalıdır. </li><li> Cerrahi bant kullanarak yatar pozisyonda fare sabitleyin. İlk cerrahi kesi yapıldı femoral arter üzerinde bir kesi yapmak. </li><li> Yaralı arter ve karşı bacak yaralanmamış arter, her ikisi için de açık açık incelemek ve femoral arter bulmak için Ekartörleri ve manyetik retraktör fiksatörler kullanarak dokuları çevreleyen sabitleyin. Periyodik sulama için tuzlu su kullanılarak dokuların nemlendirin. Steril pamuk uçlu bir aplikatör kullanılarak tuzlu su uygulayın. Arterin zarar değil dikkat edin. </li><li> Femoral arter abdominal aorta orijinal arteriotomi site izole edildikten sonra, bir segmen kravatarteriotomi orijinal sitenin yakınında ipek sütür ment. Bu dikiş femoral arter en uzak ucunu tanımlamak ve örnek işleme kolaylığı yardımcı olacaktır. </li><li> Mikro-diseksiyon makas kullanarak femoral arter çıkarılması için. Dikiş bir kesi uzak olun. Abdominal aorta yanında, femoral arter ters ucunda diğer kesi yapmak. </li><li> Tuzlu su içeren bir cam petri eksize arter aktarın. Aşırı bağ dokusu veya yağ çıkarmak için daha fazla arter disseke. Yavaşça serum fizyolojik ile durulayarak lümeninin kan çıkarın. </li><li> % 10 tamponlu formalin bir şişeye arter aktarın. Yumuşak 48 saat için sallanarak 4 ° C&#39;de şişe saklayın. </li><li> Bu histoloji için işlenene kadar saklamak için,% 70 etanol ile sabitlenmiş arter aktarın. </li><li> Parafin blok ve bölüm boyama için bloklar halinde arteri gömün. </li><li> Yaralanma ve intimal HYP boyutunu değerlendirmek için histokimyasal ve immunokimyasal boyama yapınerplasia. NOT: temsilcisi sonuçları için, biz elastik lameller ve diğer arter bileşenleri görselleştirmek için çekirdek ve genel morfoloji veya Movat en pentakrom leke görselleştirmek için hematoksilen ve eozin kullanılır. </li></ol>

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

We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire injury model with a perivascular patch is that it allows the use of genetically modified mice or murine models of disease. The endovascular manipulation of small arteries is technically demanding and this aspect has motivated the creation of models that create injury through a number of extravascular methods including ligation,11,24,25 perivascular cuffing26 and electric shock.27 In our procedures, a contralateral sham surgery can be used as a control by performing the protocol without the wire injury or, alternatively, non-operated control arteries can be used. The critical steps within the protocol are the following: (1) the surgical cut down and dissection of the femoral artery from surrounding structures, (2) the formation of the arteriotomy, (3) performance of the wire injury, (4) closure of the arteriotomy and (5) implantation of the perivascular cuff. Below we will review the major steps and address potential pitfalls in their performance. 
For the surgical cut down, the major challenge is the separation of the femoral artery from the femoral vein. Care should be taken at this stage, as it is easy to cause bleeding during the separation and the vein tears easily in comparison to the artery. Using forceps to blunt dissect and remove adventitia surround the artery and vein can help this process (see techniques shown in the video). Also, the artery may have branches underneath that can be torn if an overly aggressive technique is used. 
For the formation of the arteriotomy and wire injury, it is critical to use high quality microsurgical scissors. It is often helpful to cut at a 45-degree angle to the axis of the artery and to stabilize the cut with forceps as shown in the video. Too much or too little tension on the artery can make cutting difficult and it is sometimes helpful pull the distal tie before the proximal tie to fill the artery with blood to aid in the performing the arteriotomy. After the cut, opening the hole with the closed point of the scissors or forceps can aid in allowing entry of the wire. The size of the arteriotomy should cut approximately half way through the artery. Again, too much tension on the artery will make insertion of the wire difficult and may tear the vessel. A low to moderate level of tension should be used with the ties. When inserting the wire a slight twisting motion can be helpful but too much twisting will cause the artery to seize, preventing advancement of the wire.
A key step in performing our alternative method is the controlled local cautery that seals the vessel without blocking flow. The cautery should be heated away from the tissue and then touched to the side of the artery. Heating with the cautery while touching the vessel will burn surrounding tissues including the vein and femoral artery branches. One potential limitation of this technique is that if the arteries are not consistently cauterized there may be variations in the flow following the surgery or in the amount of inflammation caused by the burn. Analysis of the artery away from this region and consistent technique can minimize the potential impact of these limitations. We hope this alternative method will make the wire injury surgery more accessible and reduce the need for older mice for vascular injury studies.

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.

Tel yaralanma sonrasında, neointimal hiperplazi zamanla gelişir ve tipik olarak 14 ile 28 gün sonra incelenir. Şekil histolojik sonuçlar gösterildiği gibi farelerde intimal hiperplazi sağlam nesil bu iş kurşun açıklanan teknikler 3. Bir yaralanmamış femoral arter sağlam elastik lameller ve normal kalınlık ve çevresini gösterecek. Bir yaralı femoral arter intimal hiperplazi göstermek elastik lameller bozulmuş ve daha sonra zaman noktalarında yeniden endotelizasyon gösterecektir. Yeniden endotelizasyon yaklaşık 21 gün, tipik olarak tamamlanır, ancak bu, taramalı elektron mikroskobu kullanılan farelerin arka suşuna bağlıdır ve tercihen Evan mavisi ile yüz preparasyonlar en ölçülen endotel bölümlerinde işaret ya da, için immün kullanılarak değerlendirilebilir veya arterin yüz hazırlıkları tr üzerinde konfokal mikroskobu ile immün. 17,18 intimal hiperplazi ölçümü için oarterin yaralı bölgenin uzunluğu boyunca potansiyel farklılıkları hesaba yaralı arter boyunca üç bölgede intimal alanı veya intimal-to-medya oranını ölçmek için en iyisidir. <img alt="Şekil 1," src="/files/ftp_upload/52403/52403fig1highres.jpg" /> Şekil 1: femoral arter tel yaralanma için iki yöntem kritik adımlar fotoğrafları tel yaralanması gerçekleştirmek için koter yöntemiyle (alt) ligasyon yönteminin (üst) için adımlar karşılaştırılması.. Ligasyon yönteminde, kas şube sabitleyen iki dikiş önceden bağlı onlar kas şube bağlanmalarını sıkılır böylece. İki kas şube dikişlerin daha uzak sıkılır. Arteriyotomi ve tel ekleme kas dalında yapılmaktadır. Yaralanma gerçekleştirildikten sonra, kas şube kalan dikiş sıkılır ve kesilmiş. Dağlama yöntemi, sadece bir suture kas dalı altında döngü. Arteriyotomi ve tel ekleme femoral arter kas şube şube noktasında gerçekleştirilir. Yaralanma gerçekleştirildikten sonra, kesi femoral arter veya kas şube ya bağlanarak olmadan koterize edilir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52403/52403fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52403/52403fig2highres.jpg" /> Şekil 2: telli yaralanma iki tekniklerin kritik adımlardan yüksek büyütme fotoğrafları koter yöntemiyle (alt) ligasyon yönteminin (üst) için adımlar Close-up karşılaştırılması.. arteriotomi yeri kesik çizgi ile tahdit edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52403/52403fig2large.jpg" target="_blank">Bu büyük bir versiyonunu görmek için buraya tıklayınız</a>rakam. <img alt="Şekil 3," src="/files/ftp_upload/52403/52403fig3highres.jpg" /> Şekil 3:. 14 gün arterler bölümlerden prosedür histolojik sonra tel yaralanma Örnek histolojik sonuçlar, Hematoksilin ve Eosin (üst) ve Movat pentakrom boyaması en (alt) ile boyanmıştır. Resimde Etiketli lümen (L), intima (I) ve elastik lamina (EL) bulunmaktadır.

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.

Bir Perivasküler Drug Delivery Patch İmplantasyonunda ile Femoral Arter Tel Yaralanma sıçan Modeli

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

Nanyang Technological University

Research

JoVE Journal

Medicine

17.9K Görüntüleme.  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.

Biyobozunur polimerik nano partiküllerin kullanılması Tedavi Angiogenezis Programlama Kök Hücreler

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

Biyomühendislik

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.

Mikro-Bilgisayarlı Tomografi Görüntüleme için Microfil ile İskemik arka ayaklarında ve damarları bir Perfüzyon için osmotik İlaç Taşıyıcı

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

Tıp

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.

A Radyo-telemetrik Sistemi Omurilik transeksiyonu ve Embriyonik Nöral Hücre Greftler Kök Sıçanlarda Kardiyovasküler Fonksiyonu Monitör

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.

Arter Restenozun bir fare modeli: Femur Tel Yaralanma Teknik Yönleri

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.

Bir Hidrojel Construct ve Fare Miyokard Enfarktüsü Modeli Therapeutics sunun için Tutkal Yaklaşım Fibrin tabanlı.

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.

Farelerde Hızlandırılmış Ateroskleroz Indüksiyonu: "Tel-Yaralanma" Modeli

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.

Sıçan Aort veya İnferior Vena Cava de Anjiyoplasti Patch

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.

Miyokard Enfarktüsü'nün Murine Modelinde 3Boyutlu Biyobaskılı Yama Transplantasyonu

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.

Fare Karotis Arterine Manşet Tekniği ile Doku Mühendisliği Yapılmış Vasküler Greft İmplantasyonu

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.

Bir Murin Arka Bacak Modelinde Arteriyogenez Sırasında Lökosit Alımını İzlemek için Multifoton İntravital Görüntüleme

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