Bu çalışma, Bilim, Teknoloji ve G. Kore Eğitim Bakanlığı NIH hibe HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) ve Dünya Sınıfı Üniversitesi Projesi (HJ) fon tarafından desteklenmiştir.

1. Kısmi sol karotis arter ligasyonu <ol><li> Fare yaş ~ 8 hafta kullanılır. Farelerin ağırlığı en az 18 gram olması tavsiye edilir. </li><li> Boncuk enstrüman sterilizatör 30 saniyelik aletleri sterilize edin ve soğuması </li><li> Ketamin (80mg/kg) ve Xylazine (10mg/kg) intraperitoneal (ip) enjeksiyonu veya alternatif anestezi IACUC tarafından onaylanmış olarak anestezi neden. Ip enjeksiyon için, karın sol alt kadranda fare enjekte edilir. </li><li> Tamamen uyuşturuluyor kadar bir ısınma odası yerleştirin fare. Ayak tutam tepki ile anestezi derinliği olun. </li><li> Supin (karın tarafı yukarı) konumuna cerrahi bir sahnede Yeri fare. Teyp kadar ön patilerini avuç içi ve sahne aşağı arka pençeleri tek. </li><li> Mandibula ve sternum arasında boyun tüy dökücü ajan (örneğin Nair) liberal miktarda uygulayın. Yavaşça, masaj tüm saç çıkarılana kadar. </li><li> Tüy dökücü ajan temizleyin ve depilated bölgeye Betadine liberal miktarda uygulayın. </li><li> 6-0 ipek sütür ligasyonu için 1 inç uzun bölümler hazırlayın. Her fare iki adet dikiş gerekir. </li><li> , Keskin bir küçük makas kullanarak, boynun ortasında 4-6mm dikey bir kesi yapmak. Cilt ve altta yatan fasya kesilecek gerekir. </li><li> Orta hattan sola doğru hareket ettirin ve sol parotis bezi açık açık sadece sağ trakea diseksiyon başlar. Zonklayan sol karotis kolayca tespit etmek gerekir. </li><li> Kaudal karotis bifurkasyon buluncaya kadar izleyin. </li><li> Eksternal karotid arter, internal karotid arter, oksipital arter ve üstün tiroid arter, sol karotis arter kapalı aşağıdaki dört distal dallardan pervasızca açığa bifurkasyon teşrih. Bu gemiler, özellikle üstün tiroid, kırılgan olabilir. Diseksiyonu ile çok dikkatli olun. </li><li> 6-0 ipek sütür kullanarak üstün tiroid YUKARIDA eksternal karotid bağlayın. Bunu yapmak için, bir forseps arterin altından geçmek ve diğer forseps yardımı ile, önceden kesilmiş dikiş bir parça kapmak. Arterin altından geçmek için dikiş yavaşça çekin. Kravat sıkıca düğüm yakalanmış olmanın herhangi bir çevre doku kaçınarak. Etkili bir ölçüde farklı bir model tam bir ligasyonu gerçekleştirdik prosedürün sonuna kadar üstün tiroid arter altında kravat. </li><li> Internal karotid ve oksipital arterler aynı tekniği kullanarak bir düğümü ile bağlayın. </li><li> Sütür ve üstün tiroid arter açıklık konumu olun. </li><li> Doku Mend küçük bir miktar kullanarak yaklaşık cilt ve yakın cilt. </li><li> Kurtarma kadar bir ısınma odası yerleştirin fareler. Kurtarma genellikle 30 ila 60 dakika sürer. </li><li> Buprenorfin (0.1mg/kg) tek doz kullanılan anestezi, hayvanın ilk 24 saat içinde sıkıntı içinde ise, uzun süreli ağrı kesici (örn. inhalasyon izofluran) ya da eğer cerrahi sonrası hemen uygulanmalıdır. Deneyimlerimize göre, anestezi için uygulanan cerrahi, deneyimli bir cerrah tarafından yapılır ve daha fazla analjezi tabi Ketamin doz yeterli analjezi sağlar. </li></ol> 2. Karotis ultrason sınavı <ol><li> Kısmi ligasyonu akışı rahatsız kaynaklı olmadığını doğrulamak için, karotis akışı, bir gün sonrası ligasyonu muayene edilmelidir </li><li> Izofluran ile anestezi neden gerekirse anestezi indüksiyonu kutusu olabilir </li><li> Kadar görüntüleme aşamasında, göbek yan anestezi fareler yerleştirin. </li><li> Görüntüleme sahnede EKG sensör Teyp kollar ve bacaklar </li><li> Nair liberal uygulama (zaten ligasyonu işlemi yapmadı eğer bu adımı sadece ihtiyaç vardır) boyun yapraksızlaştırmayı </li><li> Rektal termometre sıcaklığı kayıt takın </li><li> Boyun bölgesine uygulayın jel Echo </li><li> B-mod başlayın. </li><li> Görüntüleme düzlemine 90 derece olmalıdır (prob üzerindeki düğme fare burun doğru işaret etmelidir) </li><li> Görüntü elde edilir kadar fare boyun prob indirin. </li><li> Aşamada sol ve sağ trakea kadar (orta hat yapısı) tanımlanır işleyin </li><li> Sol karotis arter belirleyin </li><li> Tilt görüntüleme aşamasında bir açı ve karotid görüntüleme düzlemi arasında oluşturulur. Bu açının büyük, güçlü bir Doppler sinyali olacaktır. </li><li> Place nabız dalga Doppler sol karotis ortasında. </li><li> Açı düzeltme gerektiği şekilde kullanın. </li><li> Sağ karotis ölçümler tekrarlayın. </li><li> Başarılı kısmi karotis ligasyonu diyastolde aort girişine doğru akışının ters, sağ tarafı ile karşılaştırıldığında sol karotis akış genel bir azalma (~% 80-90) neden olacaktır. </li><li> Tatmin edici görüntüler elde edildikten sonra, anestezi kapatmak, fare ve ücretsiz fare off bant res jel yankı silintraints. Fareler ısındı kurtarma odasına yerleştirin. </li></ol> 3. Karotis gelen intimal RNA izolasyonu <ol><li> Kurumsal IACUC protokole göre, CO 2 inhalasyon fareler Kurban </li><li> Bir kağıt havlu Teyp pençeleri. </li><li> Karın, göğüs kafesinin üst farenin deri kesin. </li><li> Göğüs kafesinin altında keskin bir makas çifti ile karın duvarı açın. </li><li> Diyafram forseps ile sternum kaldırın ve kesmek, daha sonra göğüs kafesi kalp maruz kesip. </li><li> Vena kava makasla kesin. </li><li> Akciğer ve karaciğer soluk hale gelene kadar sol ventrikül içinde 10 adet / mL heparin içeren serum fizyolojik ile 2 ila 3 dakika süreyle basınç serpmek (120 mmHg). </li><li> Boyun deri kesin ve karotid arterlerin maruz kadar tüm yağ, kaslar ve bağ dokuları kaldırmak. </li><li> Diseksiyon mikroskobu altında fare yerleştirin. </li><li> Ponksiyon, ikinci perfüzyon için sol karotis ligasyonu siteleri hemen altında bir delik. </li><li> ~ Sol ventrikül ile sol karotis inceledi emin olun, 10 adet / mL heparin içeren serum fizyolojik ile 1 dakika için tekrar Basınç serpmek. </li><li> Ince uçlu forseps ve küçük yaylı makas kullanılarak, karotis çevresindeki peri-adventisyal dokular dikkatle çıkarın. Bu temizleme adımı sırasında karotis sıkmak veya germek için dikkatli olun. </li><li> Kes, arkus aorta ve karotis bifurkasyon üzerinde ligasyonu noktaları arasındaki sol karotis arter. </li><li> Sağ karotis arter, sağ subklavian arter dallanma noktası ve karotid bifurkasyon arasında kesin. </li><li> Buz HBSS içeren, 35 mm kültür çanak karotis aktarın. Gerekirse, kalan peri-adventisyal doku dikkatlice çıkarın. </li><li> Her karotis başına 150 ul QIAsol lizis tamponu (Qiagen) ile doldurarak 29g bir iğne ile insülin şırınga (3 / 10 ml şırınga) hazırlayın. </li><li> Karotis bir ucunda iğne ucu dikkatlice yerleştirin. </li><li> Karotis ve iğne ucu bir forseps ile tutarken, hızlı (~ 1 sn) (intima eluat) 1.5ml bir tüpe QIAsol lizis tamponu (150 ul) temizler. </li><li> Karotis artık (medya + adventisya) bir kez HBSS, 1.5 ml tüp koymak ve sıvı nitrojen içinde ek-freeze durulayın. </li><li> İntima eluat ve donmuş artık üreticinin talimatlarına göre miRNeasy mini kiti (QIAGEN) intimal kullanarak ya da medya + adventisya RNA izolasyonu için kullanılır. </li></ol> 4. Temsilcisi Sonuçlar: Biz kısmen fare karotid arterler ve ultrason muayenesi prosedürü izleyerek bir gün gerçekleştirildi sol bağlandı. Şekil 1&#39;de gösterildiği gibi, başarılı kısmi ligasyonu kan akım hızını azaltır ve sol karotis arter diyastol sırasında akış (akış rahatsız) tersine çevirir. Yüksek yağlı diyet ile birlikte ApoE nakavt (KO) farelerde Kısmi ligasyonu, sol karotis arter (LCA) sağlam ateroskleroz neden olur, ancak sağ kontralateral karotis, iki hafta (Şekil 2). Karotis intima toplanan RNA gibi PECAM-1 gibi endotel işaretleyici genleri içeren bir-SMA (Şekil 3) medial düz kas hücre işaretleyici genlerin. <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Şekil 1" /> Şekil 1: Şematik, karotis anatomisi ve ligasyonu : Üç şube sol karotis arter (LCA) [eksternal karotid arter (ECA), internal karotis arter (ICA) ve oksipital arter (OA)] üstün tiroid arter (STA) açık bırakarak, bağlandı. <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Şekil 2" /> Şekil 2: ultrason görüntüleri akış hızı profilleri ve kısmi ligasyonu diyastolde LCA akış tersine (oklarla gösterilen) uyardığı ortaya. RCA Akış bağlanmasından sonra değişmeden kalır. <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Şekil 3" /> Şekil 3: Kısmi ligasyonu ve yüksek yağlı diyet hızla LCA ApoE KO farelerde ateroskleroz neden olur ApoE KO farelerin kısmen bağlandı ve 1 ila 3 hafta boyunca yüksek yağlı diyetle beslendi. LCA dondurulmuş bölümleri Petrol-Kırmızı-O ile boyandı. <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Şekil 4" /> Şekil 4 intimal RNA hazırlama yöntemi. Intimal RNA ve medial + adventisyal (m +) RNA sahte çalışan RCA ve LCA C57BL / 6 fareler elde edildi. RNA&#39;lar PECAM-1 ve α-SMA 18s kullanarak bir iç kontrol sistemi olarak yarı-kantitatif RT-PCR (A) ve qPCR (B, C) ​​ile analiz edildi. Çubuk grafikler, ortalama ± SEM, n = 3.

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <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></ol>

Bu yöntemde, eksternal karotid, internal karotid, oksipital arter ve üstün tiroid arter da dahil olmak üzere karotis tüm şube uygun bir şekilde tanımlanması çok önemlidir. Gösterildiği gibi ultrason kullanarak akış modeli doğrulamak için de önemlidir. Deneyimli ellerde, 500&#39;den fazla farelerin ultrason çalışmaları dayalı kısmi ligasyonu ameliyatlarında başarı oranı% 90 daha iyidir. Karotis intimal RNA hazırlıkları için, ilk perfüzyon adımdan sonra, karotis görünümünü dikkat etm...

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<td>Normal Saline (0.9% sodium chloride)</td>
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<td>Insulin syringe (3/10 ml 29g syringe)</td>
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<td>8881600145</td>
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<td>HBSS</td>
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a model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of rna isolation from carotid endothelium

Bilinen yakın ilişki olmasına rağmen, rahatsız akış aterogenez bağlayan doğrudan kanıt eksikliği olmuştur. Biz son zamanlarda, karotis kısmi ligasyonu yöntemleri değiştirilmiş bir versiyonu kullanılmıştır [1,2], akut fare karotis arterde düşük ve salınımlı akış koşulları, rahatsız akışının iki önemli özellikleri, uyardığı göstermek için. Bu modeli kullanarak, bozulmuş akışı gerçekten Apolipoprotein E nakavt fare [3] hızlı ve sağlam ateroskleroz gelişimine yol açtığı doğrudan kanıt sağladı. Ayrıca fare karotis intima [3], yüksek saflıkta endotel RNA hazırlık bir yöntem geliştirdi. Bu fare modeli ve yöntemi kullanarak, bu kısmi ligasyonu intraplak neovaskülarizasyon gibi kompleks lezyon oluşumu özellikleri ile dört hafta boyunca bir hiperlipidemik fare modeli iki hafta içinde sağlam ve hızlı bir aterom oluşumu takip, bir hafta içinde endotelyal disfonksiyon neden bulundu. Bu hızlı<em&gt; In vivo</em&gt; Model ve endotel RNA hazırlama yöntemi akış bağımlı düzenleme vasküler biyoloji ve hastalıkların altında yatan moleküler mekanizmaları belirlemek için kullanılır. Ayrıca, çalışma süresi önemli ölçüde azalır, endotelyal disfonksiyon ve ateroskleroz hedefleyen çeşitli terapötik girişimlerin test etmek için kullanılabilir.

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Bu fare karotis arter intraplak neo-vaskülarizasyon (dört hafta) ile rahatsız akış koşulları ve sonraki ateroskleroz gelişimi (iki hafta) neden olan bir kısmi karotis ligasyonu cerrahi, açıklamaktadır. Ayrıca, yüksek saflıkta endotel RNA, RNA izolasyonu, karotis intima yeni bir yöntem açıklanmaktadır.

Kısmi ligasyon Fare Karotis Arter Disturbed Akış Kaynaklı Ateroskleroz ve Karotis Endotel RNA İzolasyonu Basit Bir Yöntem Bir Model

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K Görüntüleme.  Emory University. Bu fare karotis arter intraplak neo-vaskülarizasyon (dört hafta) ile rahatsız akış koşulları ve sonraki ateroskleroz gelişimi (iki hafta) neden olan bir kısmi karotis ligasyonu cerrahi, açıklamaktadır. Ayrıca, yüksek saflıkta endotel RNA, RNA izolasyonu, karotis intima yeni bir yöntem açıklanmaktadır.

Video: A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Fare Shear stres İndüklenmiş Ateroskleroz Tetikleme Karotis Kol İmplantasyonu

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Tıp

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Sıçanlarda Common Karotid Arter interpozisyon Harici Juguler Ven Greft: Gelişmiş Mikrocerrahi Tekniği Eğitimi

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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

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

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

Restenoz Çalışmaları Karotis Arter Stent İmplantasyonu bir murin Modeli

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Fare Akciğer Entübasyonun Basit Bir Yöntem

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Yetişkin Fare Venöz Hipertansiyon Modeli: Dış Boyun Ven Anastomoza Ortak Karotis Arter.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

Karotis Arter ve Ven Juguler kateterizasyon bir Bilinçli Rat Modelinde, İnfüzyonları ve Kan Örnekleme hemodinamik Tedbirleri Gerçekleştirme

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Ferric Klorür kaynaklı Karotis Arter ve Mezenter Gemi Tromboz Fare Modeli

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Vivo Gen transferi için tavşan ortak Karotid arter endotel

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Tavşan manken dayanıklı Transgene ifade juguler ven ortak karotid arter interpozisyonu Greftler için

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...