Name: in vivo gene transfer to the rabbit common carotid artery endothelium
Uploaded: 2018-05-06T13:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

Ve destek AdVec, Inc. ve karşılaştırmalı tıp bölümü Veterinerlik Hizmetleri için cerrahi tavsiye HDAd reaktifler, Julia Feyk yönetim yardım için kullanmak için izin için teşekkür ederiz. Bu eser HL114541 ve John L. Locke, Jr. hayırsever güven tarafından desteklenmiştir.

Yazarlar ifşa gerek yok.

Cerrahi tekniğin bazı yönlerini özellikle dikkat hak. Tam pozlama ve gen transferi ve arteriotomy onarım kolaylaştırmak seferberlik ortak Karotid arter yolu ile dikkatli diseksiyon. Ancak, vasospasm önlemek için doğrudan manipülasyon Karotid arter diseksiyonu sırasında indirilmelidir. Buna ek olarak, herhangi bir arter ile bitişik kanama gazlı bezle hafif basınç uygulayarak durdurulması gerektiğini ve extravasated kan hemen normal salin alanıyla durulama ile temizlenmelidir. Ortak Karotid arter ile pa...

Bu yöntem bir transgene tavşan ortak Karotid arter endotel tanıtmak için hedeftir. Transgene getirilmesi transgene ürün biyolojik rolü değerlendirilmesi normal arter hem de insan arter hastalığı tavşan modelleri sağlar. Hastalık modelleri transgene overexpression transgene (ve protein ürün) terapötik ajanlar1,2,3,4olarak umut verici olup olmadığını ortaya çıkarabilir. CIS etkili düzenleyici elemanlarının transgene ifade kaset içinde eklenmesi damar endotel vivo içinde5,6bu öğeleri etkinliğinin değerlendirmesi sağlar. Belirli CIS etkili düzenleyici elemanlarının faaliyet bilgi daha aktif ifade kaset tasarlamak için ve büyük damar endotel vivo içinde7gen düzenlemesi mekanizmaları incelemek için kullanılabilir.
Adatavşanı are çeşitli yönlerini insan vasküler Fizyoloji ve hastalık için değerli bir model. Tavşan insanlarla birçok damar özellikleri şunlardır. Örneğin, temel hematolojik değerler, Kan durdurucu sargı yönetmelik ve vasküler boyuna gerginlik tavşan ve insanlar8arasında benzer. Tavşan modelleri vasküler hastalıklar çoğaltmak temel özellikleri de dahil olmak üzere birçok insan hastalıkları: anevrizma (benzer geometrik ve akış özellikleri)9, vasospasm (benzer tepki Endovasküler tedavi)10,11, ve ateroskleroz (intimal plaklar ile benzer özellikler bir çekirdek lipid, makrofajlar ve düz kas hücrelerinde zengin lifli bir kap içinde dahil)12,13. Buna göre tavşan modelleri tromboz, vasospasm, anevrizma, şeker hastalığı, damar grefti stenoz ve ateroskleroz8,13,14, gibi birçok vasküler hastalık gelişmiş için 15,16.
Vasküler Fizyoloji ve hastalık hayvan modelleri arasında seçme araştırmacılar için tavşan birçok avantajı vardır. Kemirgenler karşılaştırıldığında, tavşan daha büyük gemilerin kolay cerrahi işleme, Endovasküler cihazların kullanımı ve doku nicel ölçümler için daha büyük bir miktarda sağlar. Tavşan kemirgenler17daha filogenetik primatlar için çok daha yakın ve daha fazla genetik çeşitlilik outbred tavşan, daha iyi insanlar genetik değişkenliği yaklasik. Genetik çeşitlilik preklinik çalışmaları için özellikle önemli olan-genetik olarak farklı insan nüfus için uygulanan tedaviler geliştirmek için onların doğa amacı tarafından. Eğer tüm diğer birçok model türü ile tavşan genler kolayca klonlanmış veya tavşan genom ile yüksek kapsama sıralı çünkü sentez (7,48 x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway?db=oryCun2]. Diğer büyük hayvan modeller (örneğin, köpek, domuz ya da koyun) karşılaştırıldığında, tavşan satın almak nispeten ucuzdur ve ev ve onlar doğurmak ve işlemek daha kolaydır. Tavşanların her belirli vasküler hastalık modelleri bu el yazması8,12,18kapsamı dışındadır insan hastalık modelleri olarak kendi avantajları ve eksiklikleri vardır. Bir araştırmacı bu avantajları ve tavşan deneysel sorularınıza cevap için en iyi modeli olup olmadığını belirlemek için eksiklikleri gözden geçirmelidir.
Giriş endotel hücreleri vivo içinde içine deoksiribonükleik asit (DNA) düzenleyici sıralarının soruşturma bu dizilere karmaşık bir fizyolojik ortamda faaliyet sağlar. Vitro çalışmalar transfected endotel hücrelerindeki DNA düzenleyici dizileri ilk değerlendirme için yararlı olabilir; çalışmalar tekrarlanan vivo içinde5,19,20olduğunda ancak, doku kültürü modelleri seviyelerinde ifade bazen çoğaltılabilir değil. Vitro sistemleri de kültürlü vasküler hücreler arasında protein sinyal ve endotel fizyolojisi hem de iletişim temel yolları keşfetmek için yararlı olabilir; Ancak, daha karmaşık yollar veya karmaşık nüfus komşu vasküler hücre veya bağışıklık sistemi tarafından etkilenmiştir düzenleyici ağları en iyi bir vivo içinde sistem6,20dakikaya okudu. Burada açıklanan yöntemi, düzenleme çerçevesinde endotel transgene ifadesinde veya hastalık olmadan sağlam bir geminin, keşfetmek için bir platform sağlar. Vivo sistem aynı zamanda fizyolojik ve patolojik hücresel crosstalk incelenmesi ve katkıları için düzenleme gen ifade6, bağışıklık sisteminin tanımlaması izin verir.
Mikrop-line transgenesis (özellikle farelerde) transgene ifade endotel hücrelere yönetmenlik için alternatif bir yaklaşımdır. Bu yaklaşım ömür boyu transgene ifade, endotel aracılı belirli organizatörü veya düzenleyici bölgeleri21,22tarafından hedeflemede sağlayabilir. Ancak, transgenik fareler zaman alıcı ve pahalı olduğunu, birkaç transgenik hattı kez istenilen hücre tipi ve başarı yeterli transgene ifade düzeylerinin ve deneysel transgene hedefleme emin olmak için test gerekir fare sistemleri sonuçlarında zorlanma bağımlı olabilir. Endotel hedefli transgenes ile fare transgenik modelleri pek çok avantajı var: cerrahi transgenesis elde etmek için her deneysel hayvan üzerinde gerçekleştirmek için gerek yoktur, deneysel fareler çok sayıda diğer kullanılabilir transgenik farelerde ile yetiştirilmiş genetik ve fenotipik etkileşimleri test etmek için sipariş ve fenotipleri karakterizasyonu kolaylaştırmak fare proteinler ile tepki antikorların geniş bir seçim. Ancak, genellikle transgenes germ hattı üzerinden endotel için hedefleme transgene ifade damarlara, hangi transgene ürün etkili olan siteyi belirlemek üzere22 boyunca sonuç. Transgene ürün salgılanan, damarlara boyunca endotel hücreleri tarafından salgılanan bir transgene ürün içerisindeki bir hayvan sitelerin her noktada biyolojik aktivitesi olabilir çünkü bu özellikle doğrudur. Bu el yazması anlatılan yöntem teknik uzmanlık ve özel İmkanlar gerektirir, ancak daha az zaman alıcı ve bir özel endotel transgenik fare çizgi geliştirme daha ucuz olabilir. Bu işlev, bir kesim büyük arterin endotel hücreleri seçerek bir proteinin değerlendirilmesi için sağlar ve kontralateral ortak karotis kullanımına (deneysel arasında değişebilir sistemik faktörleri ortadan eşleştirilmiş denetimi olarak izin verir hayvanlar-Örneğin, kan basıncı veya kolesterol düzeyleri-kontrolsüz değişkenler olarak).
Gen tedavisi damar hastalıklarının tedavisi için umut verici bir yaklaşımdır özellikle kronik hastalıklar, çünkü tek bir uygulama sürekli ya da büyük olasılıkla ömür boyu ifade bir tedavi gen23sağlayabilir. Gen tedavisi tedavi vaadi somatik gen transferi, sık sık kan yoluyla birçok viral vektörler hepatotropic çünkü nispeten kolay bir hedef olan karaciğer24,25, hedefleme hayvan modellerinde keşfedilmeyi. Ancak, damar hastalıkları üzerinde etkisi olduğu için karaciğere hedef gen tedavisi sistemik overexpression proteinlerin elde etmek gerekir. Bu genellikle büyük dozlarda toksik veya hatta ölümcül26olabilir vektör gerektirir. Ayrıca, sistemik protein zam hedef kapalı yan etkileri, karmaşık veya hatta deneysel sonuçlar yorumlanması karanlık riskini düzeyde arttı. Çünkü infüzyon vektör yaygın olarak transduced Arteryel segment Dissemine değil ve yerel vasküler etkileri olmadan elde edilebilir bu el yazması açıklandığı gibi vasküler endotel hedefleme yerel gen tedavisi sistemik yan etkileri önlemek olabilir Sistemik plazma düzeyleri protein değişimler. 27 vektör çok daha düşük bir miktarda Ayrıca, sağlam hepatik iletim ulaşmak için gerekenden bir Arteryel kesimini transduce gerekiyordu. Karaciğer ifadeden transgene üst düzey transgene ifade saklanması için ise tekrarlanan dozaj gerektiren büyük olasılıkla hücre ciro nedeniyle, zaman içinde azalmaya bildirilmiştir. 28 buna ek olarak, istikrarlı ifade en az 48 hafta chow beslenen tavşanların ve en az 24 hafta kolesterol beslenen tavşanların aterosklerotik lezyonları içinde endotel düşük devir hızı sağlar. 1 , 27
Bu yöntemi, gen transferi tavşan ortak Karotid endotel için uygun olup olmadığını belirlemek için özel araştırma hedefleri bağlamında avantajları ve dezavantajları (Tablo 1) dikkate alınmalıdır. Bu yöntemin avantajları şunlardır: outbred adatavşanı are insan genetik çeşitliliğin daha iyi temsilcisi daha doğuştan fareler (preklinik iş için önemli); Tavşan daha kolay manipülasyon için daha büyük gemiler ve daha fazla doku analizi için sağlar; yöntem endotel hedefli transgene ifade mikrop-line transgenik farelerde hedefleme endotel göründüğünden çok daha hızlı bir şekilde elde edebilirsiniz; vektör doz kolaylıkla modeli çeşitli düzeyleri transgene ifade için ayarlanabilir; büyük damar endotel için belirli işlemleri soruşturma olması; ve yerel vasküler transgenesis ters karotis aynı hayvan kontrolsüz değişkenler olarak sistemik faktörleri ortadan bir denetim olarak kullanılmak üzere izin verir. Dezavantajları şunlardır: Özel İmkanlar ve uzmanlık gereklidir; daha az genetiği değiştirilmiş arka plan hangi deneme tavşan farelerde kullanılabilir; ve tavşan fare proteinler (için immunodetection transgene protein ve deneysel sonuçlar yorumlama önemli olabilir diğer antijenleri) karşı antikorlar daha az kapsamlı bir seçki.

<table><tbody><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3mL syringe with 24G needle</td><td>Becton Dickinson</td><td>309571</td><td>2x for gene transfer surgery; 3x for harvest surgery</td></tr><tr><td>1mL syringe with 27G needle</td><td>Becton Dickinson</td><td>309623</td><td>6x for gene transfer surgery; 1x for harvest surgery</td></tr><tr><td>20mL syringe, luer lock</td><td>Nipro Medical Corp</td><td>JD+20L</td><td></td></tr><tr><td>Catheters, 24G x 3/4&quot;</td><td>Terumo Medical Products</td><td>SROX2419V</td><td></td></tr><tr><td>19G needle</td><td>Becton Dickinson</td><td>305187</td><td>Gene transfer surgery only</td></tr><tr><td>21G needle</td><td>Becton Dickinson</td><td>305165</td><td>For 20 mL syringe of saline</td></tr><tr><td>Gauze 4&quot; x 4&quot;</td><td>Dynarex</td><td>3242</td><td>~10-15 per surgery</td></tr><tr><td>3-0 silk suture</td><td>Covidien Ltd.</td><td>S-244</td><td></td></tr><tr><td>5-0 silk suture</td><td>Covidien Ltd.</td><td>S-182</td><td>Gene transfer surgery only</td></tr><tr><td>7-0 polypropylene suture</td><td>CP Medical</td><td>8648P</td><td>Gene transfer surgery only</td></tr><tr><td>5-0 polyglycolic acid suture</td><td>CP Medical</td><td>421A</td><td>Gene transfer surgery only</td></tr><tr><td>3-0 polyglycolic acid suture</td><td>CP Medical</td><td>398A</td><td>Gene transfer surgery only</td></tr><tr><td>Alcohol swabs</td><td>Covidien Ltd.</td><td>6818</td><td>For placement of I.V. line</td></tr><tr><td>Catheter plug</td><td>Vetoquinol</td><td>411498</td><td>Gene transfer surgery only</td></tr><tr><td>Ketamine HCl, 100 mg/mL</td><td>Vedco Inc.</td><td>05098916106</td><td></td></tr><tr><td>Xylazine, 100 mg/mL</td><td>Akorn Inc.</td><td>4821</td><td></td></tr><tr><td>Lidocaine HCl, 2%</td><td>Pfizer</td><td>00409427702</td><td></td></tr><tr><td>Bupivacaine HCl, 0.5%</td><td>Pfizer</td><td>00409161050</td><td></td></tr><tr><td>Beuthanasia D-Special</td><td>Intervet Inc.</td><td>NDC 00061047305</td><td>Harvest surgery only</td></tr><tr><td>Buprenorphine HCl, 0.3 mg/mL&amp;nbsp;</td><td>Patterson Veterinary</td><td>12496075705</td><td>Gene transfer surgery only</td></tr><tr><td>Saline IV bag, 0.9% sodium chloride</td><td>Baxter</td><td>2B1309</td><td>2x for gene transfer surgery; can use vial of sterile saline in place of one</td></tr><tr><td>Heparin&amp;nbsp; (5000 U/mL)</td><td>APP Pharmaceuticals</td><td>NDC 63323-047-10</td><td>Gene transfer surgery only</td></tr><tr><td>Fentanyl patch, 25 mcg/hr&amp;nbsp;</td><td>Apotex Corp.</td><td>NDC 60505-7006-2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Gene transfer vector</td><td></td><td></td><td>Dilute 350 &amp;micro;L per artery; 2 x 10&lt;sup&gt;11&lt;/sup&gt; vp/mL for adenovirus; gene transfer surgery only</td></tr><tr><td>&lt;strong&gt;Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metzenbaum needle holder 7&quot; straight</td><td>Roboz</td><td>RS-7900</td><td>Gene transfer surgery only</td></tr><tr><td>Operating scissors 6.5&quot; straight blunt/blunt</td><td>Roboz</td><td>RS-6828</td><td></td></tr><tr><td>Needle holder /w suture scissors</td><td>Miltex</td><td>8-14-IMC</td><td>Gene transfer surgery only</td></tr><tr><td>Castroviejo scissors</td><td>Roboz</td><td>RS-5658</td><td></td></tr><tr><td>Castroviejo needle holder, 5.75&quot; straight with lock</td><td>Roboz</td><td>RS-6412</td><td>Gene transfer surgery only</td></tr><tr><td>Stevens scissors 4.25&quot; curved blunt/blunt</td><td>Roboz</td><td>RS-5943</td><td></td></tr><tr><td>Alm retractor 4&quot; 4X4 5mm blunt prongs</td><td>Roboz</td><td>RS-6514</td><td>2x</td></tr><tr><td>Backhaus towel clamp 3.5&quot;</td><td>Roboz</td><td></td><td>4x</td></tr><tr><td>Micro clip setting forceps 4.75&quot;</td><td>Roboz</td><td>RS-6496</td><td>Gene transfer surgery only</td></tr><tr><td>Micro vascular clips, 11 mm</td><td>Roboz</td><td></td><td>2x for gene transfer surgery only</td></tr><tr><td>Surg-I-Loop</td><td>Scanlan International</td><td>1001-81M</td><td>5 cm length</td></tr><tr><td>Bonaccolto forceps, 4&amp;rdquo; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td><td>Roboz</td><td>RS-5210</td><td></td></tr><tr><td>Dumont #3 forceps Inox tip size .17 X .10mm</td><td>Roboz</td><td>RS-5042</td><td></td></tr><tr><td>Graefe forceps, 4&amp;rdquo; (10 cm) long serrated straight, 0.8mm tip</td><td>Roboz</td><td>RS-5280</td><td></td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; straight, 1.3mm tips</td><td>Roboz</td><td>RS-7110</td><td>2x</td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; curved, 1.3mm tips</td><td>Roboz</td><td>RS-7111</td><td></td></tr><tr><td>Jacobson mosquito forceps 5&quot; curved extra delicate, 0.9 mm tips</td><td>Roboz</td><td>RS-7117</td><td></td></tr><tr><td>Kantrowitz forceps, 7.25&quot; 90 degree delicate, 1.7 mm tips</td><td>Roboz</td><td>RS-7305</td><td></td></tr><tr><td>Tissue forceps 5&quot;, 1X2 teeth, 2 mm tip width</td><td>Roboz</td><td>RS-8162</td><td></td></tr><tr><td>Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td><td>Fine Science Tools</td><td>11092-12</td><td>2x</td></tr><tr><td>Adson forceps, 12 cm, serrated, straight</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Veterinary electrosurgery handpiece and electrode</td><td>MACAN Manufacturing</td><td>HPAC-1; R-F11</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Suite Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Circulating warm water blanket and pump</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Forced air warming unit</td><td>3M</td><td>Bair Hugger Model 505</td><td>Gene transfer surgery only</td></tr><tr><td>IV infusion pump</td><td>Heska</td><td>Vet IV 2.2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane vaporizer and scavenger</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Veterinary multi-parameter monitor</td><td>Surgivet</td><td>Surgivet Advisor</td><td></td></tr><tr><td>Veterinary electrosurgery unit</td><td>MACAN Manufacturing</td><td>MV-9</td><td></td></tr><tr><td>Surgical microscope</td><td>D.F. Vasconcellos</td><td>M900</td><td>Needs ~16x magnification</td></tr></tbody></table>

in vivo gene transfer to the rabbit common carotid artery endothelium

Bu yöntem bir transgene izole segmentlerinin her iki tavşan ortak Karotid arter endotel tanıtmak için hedeftir. Böylece bir dedektif endotel ifade transgenes biyolojik rolleri belirlemek ve DNA dizilerinin büyük arter içinde vivo transkripsiyon etkinliği ölçmek için izin veren odak seçici endotel transgenesis yöntemi elde endotel hücreleri. Bir transgene ifade viral vektör Arteryel lumen teslim için tavşan ortak Karotid arter ve bir arteriotomy cerrahi yalıtım yöntemini kullanır. Lümen vektörde kısa kuluçka süresi Lümen içindekiler sonraki aspirasyon ile tespit iletim veya ifade dışında endotel transgene verimli ve dayanıklı ifade ulaşmak için yeterli izole arter kesimi. Yöntemi biyolojik etkinlikler transgene ürünlerin değerlendirilmesi normal arterler ve modelleri insan damar hastalığının gen teslim (örn. diğer sitelere hedefleme tarafından kaynaklanmış olabilir sistemik etkileri kaçınırken sağlar karaciğer) veya genetik yapıları için endotel germ hattı transgenesis tarafından teslim alternatif bir yaklaşım. Uygulama yönteminin yetenekli bir cerrah ve anestezi, iyi donanımlı ameliyathane, satın alma ve konut tavşan maliyetlerinin ihtiyacını ve uzmanlık gen transferi vektör yapım ve kullanım için ihtiyaç ile sınırlıdır. Bu yöntem ile elde edilen sonuçlar içerir: transgene ile ilgili değişiklikler arter yapısı, cellularity, hücre dışı matriks veya vasomotor işlevi; artar veya damar iltihabı indirimleri; vasküler hücre apoptosis içinde değişiklikler; ve ilerleme, geriliği veya regresyon intimal Hiperplazi veya ateroskleroz gibi hastalıkların. Yöntemi aynı zamanda doğal ve sentetik DNA düzenleyici diziler transgene ifade içeren sonuçları sağlayan endotel hücrelerinde değiştirme olanağı ölçümü sağlar: transgene düzeyde mRNA, transgene protein düzeyleri ve düzeyleri transgene enzimatik aktivite.

Güven ile bu yöntemi uygulamak için ön deneyler işleci luminal endotel hücreleri ifadede öncelikle transgene ile verimli ve tekrarlanabilir gen transferi elde kurmak gereklidir. Deneyim, bu en kolay β-galaktozidaz ifade eder bir vektör kullanarak değerlendirilir. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) ortak Karotid segmentlerinin boyama 3 gün sonra vektör infüzyon, hem de nicel ters transkripsiyon Polimeraz zincir ile β-galaktozidaz mRNA (mesajcı RNA) öl...

Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

Bu yöntem bir transgene tavşan Karotid arter endotel tanıtmak için hazırlanmıştır. Transgene getirilmesi transgene ürün normal damar veya hastalık modelleri biyolojik rolü değerlendirmesini sağlar. Yöntem düzenleyici DNA dizilerinin etkinliği ölçmek için yararlıdır.

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 ...

in-vivo-gene-transfer-to-the-rabbit-common-carotid-artery-endothelium

Research

JoVE Journal

Medicine

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

9.0K Görüntüleme.  University of Washington. Bu yöntem bir transgene tavşan Karotid arter endotel tanıtmak için hazırlanmıştır. Transgene getirilmesi transgene ürün normal damar veya hastalık modelleri biyolojik rolü değerlendirmesini sağlar. Yöntem düzenleyici DNA dizilerinin etkinliği ölçmek için yararlıdır.

Video: In Vivo Gene Transfer to the Rabbit Common Carotid Artery 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

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

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 ...

Tıp

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Ortak Bozuklukları de Novo Mutasyonlar, otizm ve şizofreni gibi tanımlayın Bir Strateji

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Klinik öncesi Model İskemik Kalp Yetmezliği Gen Transferi

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Hidrolize Çapraz bağlayıcılar yoluyla Adenoviral vektörler gergin kullanma Metalik Stent Yüzeylerden Vasküler Gen Transferi

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Tavşan modeli hızlandırılmış ateroskleroz: iliyak arter balon yaralanma bir metodolojik açıdan

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

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, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Diyabetik tavşanların arka ekstremite Iskemi preklinik modeli

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Arteriyel Kan Basıncı Altında İntimal Hiperplazi değerlendirmek için Ven Greftleri kullanarak Revaskülarizasyon Cerrahisi Taklit Bir Tavşan Venöz Interposition Modeli

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

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 ...

Biyomühendislik

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model

Source: Nishio, H., et al. <a href="https://www.jove.com/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
This video demonstrates the interposition of the rabbit carotid artery with the jugular vein to induce venous intimal hyperplasia by subjecting the veins to arterial pressure. This model will help investigate molecular and genetic interventions to attenuate the progression of intimal hyperplasia after revascularization surgeries.

Karotis Arter ve Juguler Ven İnterpozisyonu: Tavşan Modelinde Venöz İntimal Hiperplaziyi İndüklemek İçin Bir Teknik

Source: Nishio, H., et al. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under ...