Bu çalışma CKB için NIH (RO1 HL098228) bir hibe, kısmen desteklendi.

NOT: Bütün hayvan prosedürleri Nationwide Çocuk Hastanesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1.. Greft İmalat <ol><li> Bir davlumbaz altında 2 ml dioksan içerisinde 100 mg P (LA / CL) eklenerek ε-kaprolakton ve L-laktid kopolimeri P (LA / CL) çözeltisi olun. Bir girdap çözüm yerleştirin ve tamamen erimesi için 1-1.5 saat boyunca sürekli olarak karıştırınız. </li><li> Bu arada, (PGA) dondurucu hissettim poliglikolik asit tabakasını kaldırmak ve birkaç 5 x 8 mm bölümleri kesip. Ayrıca sadece filtrenin üstünde bir 0.1-10 ul pipet ucu kesilmiş. </li><li> Bir pipet ucunun uzak ucunda bir 19 G iğne (1.5 uzunluk) takın ve PGA mikro forseps kullanılarak iğnenin keçe sarın. </li><li> 19 G iğne içine takıldığında ise dikkatlice, lümen bir kör 18 G iğne kullanılarak, yakın kısmı daha düz bir pipet ucu, distal ucuna keçeyi itin. </li><li> Pipet40 ul P (CL / LA) üst ucu içine pipet çözeltisi. PGA çözümü ile hissetti doyurabilecek. Sonra pipet dağıtıcı kullanarak hava kabarcıkları dışarı itmek. Gerekirse bu işlemi tekrarlayın. </li><li> Yer, 50 ml bir tüp içinde greft ve 20 dakika boyunca -80 ° C dondurucu içine yerleştirin. Iğnelerin baş aşağı doğru karşı karşıya olduğundan emin olun. </li><li> Bir liyofilizerde içine tüp transferi ve 24 saat için vakum. Hava akımı oluşturacak şekilde tüpün kapağını açmak için emin olun. </li><li> Greft dışarı atın ve iğnelerden çıkarın. A ~ 5 mm bölümünü terkeden greft iki ucunu kesme ve şeklini korumak için, iğnelerin üzerine geri koyun. Desikatörde greft tutun. </li><li> Bir biyogüvenlik kaput O / N önce hücre tohumlama UV ışık altında iskele yerleştirin. </li></ol> 2.. Kemik İliği Mononükleer Hücre Hasat ve İzolasyon <ol><li> Ketamin / ksilazin aşırı dozda (ketamin 200 mg / kg ketamin hidroklorid ve 20 mg / kg) ile fareler Euthanize. </li><li> Kaldır kemikler (femur bir10 cc RPMI sahip bir Petri kabındaki 10 greft implantasyonu ve yer 3 fareden d tibias). 3 cc RPMI ile yeni bir Petri kabı, bir 25 G iğnesi olan bir şırınga kullanılarak, kemik ve kemik iliği gömme iki ucunu kesin. 15 cc tüpte kemik iliği ve RPMI çözeltisi toplayın ve BM arasında kalan toplamak için ek 2 cc RPMI ile petri yıkayın. </li><li> Örnek (5-10 ul) alın ve bir otomatik hücre sayıcı veya hemasitometre kullanarak hücre saymak. Sonucu kaydedin. </li><li> 15 cc santrifüj tüpüne 5 cc Ficoll koyun ve kemik iliği ve RPMI çözüm ekleyin. Ficoll&#39;ün ile karıştırma önlemek için çok nazikçe çözüm ekleyin. </li><li> 24 ° C&#39;de &quot;HAYIR FRENİ&quot; ile 30 dakika boyunca 528 x g&#39;de santrifüj </li><li> Üst pembe katmanı kaldırın. MNC katmanı orta berrak tabaka Şekil 1, toplayın, ve PBS 01:01 ile sulandırmak. </li><li> 24 ° C&#39;de 10 dakika boyunca 528 x g&#39;de santrifüj solüsyonu inceltilmiş MNC </li><li> Süpernatantı ve di kaldırud 5 ml PBS ile pelet. </li><li> 24 ° C&#39;de 10 dakika boyunca 528 x g&#39;de topak çözeltisi santrifüj </li><li> Süpernatant kaldırmak. RPMI (~ 200 ul) uygun miktarda pelet seyreltin. </li><li> 5-10 ul örnek almak ve bir otomatik hücre sayıcı veya hemasitometre kullanarak hücreleri saymak. Sonucu kaydedin. Bir kez daha sayım hücreyi tekrarlayın ve ortalama hücre sayısını hesaplamak. </li><li> RPMI kullanarak ul 1.000.000 cells/10 hücre konsantrasyonu sulandırmak. </li></ol> 3.. Hücre Serpilmesi <ol><li> 5 dakika boyunca luminally 5 ul RPMI ilave edilerek ön ıslatılmış iskele, sonra RPMI çıkarın. </li><li> Iskele lümen Adım 2.12 RPMI 10 ul kemik iliğinden elde edilen mono nükleer hücreleri ekleyin ve 10 dakika hücreleri iskele üzerine takmak için izin için bekleyin. </li><li> 1 cm uzunluğunda bir 19 G iğne Kesme ve iskele şeklini korumak için, skafoldun lümeni içine iğne koydu. Bir 24-çukurlu plaka içindeki örnek yerleştirin. &lt;/ Li&gt; <li> Her kuyuya 1.000 ul RPMI ilave edin ve bir kuluçka makinesi içinde O / N inkübe edin. </li></ol> 4. Greft İmplantasyonu <ol><li> Ameliyat öncesi tüm cerrahi aletler otoklava sokun: ince makas, 3x mikro forseps, 2x mikro vasküler kelepçeler, 1x kelepçe uygulayarak forseps, 1x mikro iğne tutucu, 1x yaylı makas, 1x ekartörünü 1x. </li><li> 6-8 haftalık dişi C57BL / 6 doku mühendisliği damar grefti alıcı olarak kullanılır. Kafesinden fare çıkarın ve ağırlık, daha sonra bir ketamin / ksilazin kokteyl (ketamin 100 mg / kg ketamin hidroklorid ve 10 mg / kg) ile karnın alt sağ çeyrek dairesi içine bir intraperitoneal enjeksiyon yoluyla anestezi. Ketoprofen (5 mg / kg, IP) bir ön anestezi analjezik olarak kullanılır. </li><li> Masal sıkıştığı tarafından sedasyon seviyesini kontrol edin, sonra karın saç klibi. Steril Oftalmik merhem ile gözleri yağlayın ve bir yastık üzerinde bir dorsal recumbence pozisyonda fare yerleştirin. Betadine ve alkol pedleri ile karın dezenfekte edin. Costeril bir örtü ile fareyi ver ve sadece kesi alanı açığa. </li><li> Suprapubik bölgeye xiphoid aşağıdan bir orta hat laparotomi kesi yapmak ve bir öz-istinat ekartörünü yerleştirin. Tuzlu çözelti ile nemlendirilmiş gazlı bez bağırsakları sarın. Açık açık infrarenal aorta ve vena kava tanımlar. </li><li> Proksimal ve aort ve vena kava distal taraflarında iki mikro vasküler kelepçeler yerleştirin sonra açık açık vena kava transekt vena kava gelen aort ayrı. Gerekirse, konik iğne üzerinde 10-0 monofilament sütür ile abdominal aort dalları Arter. </li><li> Steril 10-0 sütür kullanılarak anastomoz sonuna yakın ve uzak ucu ile alt vena kava interpozisyonundan greft dikiniz. Fare anatomisine bağlı greft, genellikle 1-2 mm, Trim. Proksimal ve distal uçlarında hem bir tane dikiş greft elde edin ve greft diğer taraftan 4-5 stiches sürekli dikiş başlar. Ön tarafı bitirdikten sonra, kelepçeler ve greftler çevirmekdiğer tarafa ve greft arka tarafını dikin. Implantasyon sırasında, akut trombozu önlemek için sık sık heparin solüsyonu ile greft yıkayın. </li><li> Proksimal kelepçesini çıkarın ve bir topikal emilebilir steril hemostat ajanı uygulayarak kanamayı kontrol. Kanama tamamen durduğunda, distal kelepçesini çıkarın ve kanama aynı şekilde kontrol. Greft içinden emin kan akışları olun. </li><li> Dahil dişli bir iğne ile 6-0 siyah bir poliamid monofilament dikiş ile iki kat olarak abdominal kas ve deri kapatın. </li><li> 0.5 ml serum fizyolojik subkutan enjekte edilir ve fare tamamen mobil kadar bir ısınma pad üzerinde bir kurtarma kafes fare yerleştirin. Kurtarma üzerine, kağıt yatak ile yeni kafesine fareyi dönmek. 48 saat boyunca ağrı ilaç (ibuprofen, 30 mg / kg, içme suyu) ver. </li></ol>

<ol>
	<li>Heart Association, A. m. e. r. i. c. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics&#8212;2012+Update.">Heart Disease and Stroke Statistics&#8212;2012 Update.</a> Circulation. 125, (2012).</li><li>Shinoka, 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=Creation+Of+Viable+Pulmonary+Artery+Autografts+Through+Tissue+Engineering.">Creation Of Viable Pulmonary Artery Autografts Through Tissue Engineering.</a> The Journal of Thoracic and Cardiovascular Surgery. 115, 536-546 (1998).</li><li>Hibino, N., 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=Late-term+results+of+tissue-engineered+vascular+grafts+in+humans.">Late-term results of tissue-engineered vascular grafts in humans.</a> The Journal of Thoracic and Cardiovascular Surgery. 139, 431-436 (2010).</li><li>Shin'oka, 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=Midterm+clinical+result+of+tissue-engineered+vascular+autografts+seeded+with+autologous+bone+marrow+cells.">Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 129, 1330-1338 (2005).</li><li>Brennan, M. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+autologous+tissue-engineered+venous+conduit+from+bone+marrow-derived+vascular+cells:+optimization+of+cell+harvest+and+seeding+techniques.">Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques.</a> Journal of Pediatric Surgery. 42, 198-202 (2007).</li><li>Hibino, N., 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=Tissue-engineered+vascular+grafts+form+neovessels+that+arise+from+regeneration+of+the+adjacent+blood+vessel.">Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel.</a> The FASEB Journal. 25, 2731-2739 (2011).</li><li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+macrophages+in+neovessel+formation+and+the+development+of+stenosis+in+tissue-engineered+vascular+grafts.">A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts.</a> The FASEB Journal. 25, 4253-4263 (2011).</li><li>Naito, Y., 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=Characterization+of+the+Natural+History+of+Extracellular+Matrix+Production+in+Tissue-Engineered+Vascular+Grafts+during+Neovessel+Formation.">Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation.</a> Cells Tissues Organs. 195, 60-72 (2012).</li><li>Naito, Y., 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=Beyond+Burst+Pressure:+Initial+Evaluation+of+the+Natural+History+of+the+Biaxial+Mechanical+Properties+of+Tissue+Engineered+Vascular+Grafts+in+the+Venous+Circulation+Using+a+Murine+Model.">Beyond Burst Pressure: Initial Evaluation of the Natural History of the Biaxial Mechanical Properties of Tissue Engineered Vascular Grafts in the Venous Circulation Using a Murine Model.</a> Tissue Eng. 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L., 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=Tissue-engineered+arterial+grafts:+long-term+results+after+implantation+in+a+small+animal+model.">Tissue-engineered arterial grafts: long-term results after implantation in a small animal model.</a> Journal of Pediatric Surgery. 44, 1127-1133 (2009).</li><li>Lee, Y. U., Naito, Y., Kurobe, H., Breuer, C. K., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biaxial+mechanical+properties+of+the+inferior+vena+cava+in+C57BL/6+and+CB-17+SCID/bg+mice.">Biaxial mechanical properties of the inferior vena cava in C57BL/6 and CB-17 SCID/bg mice.</a> Journal of Biomechanics. 46, 2277-2282 (2013).</li></ol>

Yazarlar ifşa hiçbir şey yok.

TEVG fare modeli neotissue stenoz oluşumu ve geliştirilmesi hücresel ve moleküler mekanizmaları incelemek için değerli bir araçtır. Tohumlanmış BM-MNC greft 11 tohumlanmış hücreleri hem histolojik ve SEM görüntüleri gösterilmiştir. Hücre Ekme etkinliği, bir DNA deneyi 7 ile gösterilmiştir. Bu model sistemi kullanarak biz cep tohumlama insan klinik deneme 3 başarısızlık birincil modu oldu TEVG stenoz, gelişiminin insidansını azalttığını göstermiştir. To...

Konjenital kalp kusurları ABD&#39;de canlı doğumların yaklaşık% 8 etkileyen ciddi durumlardır. Konjenital kalp kusurları veya 2.4 1.000 canlı doğum olan bebeklerin yaklaşık% 25&#39;i, hayatlarının 1 ilk yıl invaziv tedavi gerektirir. Konjenital kalp hastalığı için en etkili tedavi rekonstrüktif cerrahi olduğunu. Ne yazık ki, mevcut vasküler kanalların kullanımından kaynaklanan komplikasyonlar ameliyat sonrası morbidite ve mortalitenin en önemli nedenidir. Bu sorunu çözmek için, klinik kullanım 2 için ilk doku mühendisliği vasküler greft (TEVGs) geliştirdi. TEVGs otolog kemik iliğinden elde edilmiş tek-çekirdekli hücreleri (BM-MNC) ile tohumlandı ve konjenital kalp ameliyatı venöz kanalları olarak implante edilen biyolojik olarak parçalanabilir polyester tüplerden inşa edilmiştir. Sonuçlar takipte 1-3 yıldır mükemmel açıklık oranları gösterdi, ama darlığının önemli insidansı <s&gt; 3,4 kadar. Bu vasküler neotissue oluşumu ve TEVG darlık gelişimini altında yatan mekanizmanın daha iyi anlaşılması gerektiği açıktı. Daha TEVGs gelişimini ve darlık gelişme mekanizmasını anlamak için, bir koyun bir model 5,6 oluşturuldu. Bu modelde, TEVGs başarılı bir şekilde canlı damarları içine transforme edildi ve morfolojisi ve ana damarlar fonksiyonu hem de benzerdi. Büyük bir hayvan modelinin bu kullanımı TEVGs klinik kullanımını destekli önemli klinik öncesi bilgi veren iyi bir ilk adım oldu. Ancak, büyük hayvan modelleri kullanılarak TEVGs vasküler neotissue oluşumu hücresel ve moleküler mekanizmaları tam anlaşılması nedeniyle türlerin spesifik moleküler araçlar eksikliği nedeniyle vasküler hücre fenotipleri moleküler karakterizasyonu kısıtlamalar sınırlıdır. Bu eksikliklerin üstesinden gelmek için, TEVGs bir murin modeli fare genetik hızlı ilerleme ve geniş Molecula nedeni ile geliştirilmiştirdaha kısa bir zaman ölçek ilave avantajı ile r karakterizasyonu. Sıçangil modeli IVC araya girme sadık büyük hayvanlarda ve insanlarda meydana neoveseller oluşum sürecini değinmeyecek ama çok daha kısa bir zaman süreci boyunca 6-9. Burada, biyolojik olarak parçalanabilir yapı iskelesi kullanılarak küçük ölçekli aşı üretimi için ayrıntılı bir protokol, BM-MNC hasat ve izolasyon, bir platform üzerinde BM-MNC tohumlama ve bir murin modelinde greft tarif edilmiştir.

<table><tbody><tr><td>Polyglycolic acid (PGA) felt</td><td>Biomedical Structures</td><td></td><td>Custome ordered</td></tr><tr><td>Pipet tip, 0.1-10 &amp;mu;l&amp;nbsp;</td><td>Fisher Sientific</td><td>02-707-456</td><td></td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>Labconco</td><td>7070020</td><td></td></tr><tr><td>RPMI medium 1604</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Petri dish</td><td>BD</td><td>353003</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>15 cc tube&amp;nbsp;</td><td>BD</td><td>352096</td><td></td></tr><tr><td>Ficoll</td><td>Sigma</td><td>10831-100ml</td><td>Also called &#039;Histopaque&#039;</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Littauer bone cutter 4.5&quot; Straight</td><td>Roboz</td><td>RS-8480</td><td>For BM harvesting</td></tr><tr><td>Forceps 4.5&quot;</td><td>Roboz</td><td>RS-8120</td><td>For BM harvesting</td></tr><tr><td>Scissors 4.5&quot;</td><td>Roboz</td><td>RS-5912</td><td>For BM harvesting</td></tr><tr><td>Microscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>C57BL/6J (H-2b), Female</td><td>Jackson Laboratories</td><td>664</td><td>8-12 weeks</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Hospira Inc.</td><td>NDC 0409-2053</td><td></td></tr><tr><td>Xylazine sterile solution</td><td>Akorn Inc.</td><td>NADA# 139-236</td><td></td></tr><tr><td>Ketoprofen</td><td>Fort Dodge Animal Health</td><td>NDC 0856-4396-01</td><td></td></tr><tr><td>Ibuprofen</td><td>PrecisionDose</td><td>NDC 68094-494-59</td><td></td></tr><tr><td>Heparin sodium</td><td>Sagent Pharmaceticals</td><td>NDC 25021-400</td><td></td></tr><tr><td>Saline solution (sterile 0.9% sodium chloride)</td><td>Hospira Inc.</td><td>NDC 0409-0138-22</td><td></td></tr><tr><td>0.9% Sodium chloride injection</td><td>Hospira Inc.</td><td>NDC 0409-4888-10</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Iodine prep pads</td><td>Triad Disposables, Inc.</td><td>NDC 50730-3201-1</td><td></td></tr><tr><td>Alcohol prep pads</td><td>McKesson Corp.</td><td>NDC 68599-5805-1</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific</td><td>23-400-118</td><td></td></tr><tr><td>Fine scissor</td><td>FST</td><td>14028-10</td><td></td></tr><tr><td>Micro-adson forcep</td><td>FST</td><td>11018-12</td><td></td></tr><tr><td>Clamp applying forcep</td><td>FST</td><td>00072-14</td><td></td></tr><tr><td>S&amp;amp;T Vascular clamp</td><td>FST</td><td>00396-01</td><td></td></tr><tr><td>Spring scissors</td><td>FST</td><td>15008-08</td><td></td></tr><tr><td>Colibri retractors</td><td>FST</td><td>17000-04</td><td></td></tr><tr><td>Dumont #5 forcep</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Dumont #7 - fine forceps</td><td>FST</td><td>11274-20</td><td></td></tr><tr><td>Dumont #5/45 forceps</td><td>FST</td><td>11251-35</td><td></td></tr><tr><td>Tish needle holder/forceps</td><td>Micrins</td><td>MI1540</td><td></td></tr><tr><td>Black polyamide monofilament suture, 10-0</td><td>AROSurgical Instruments Corporation</td><td>TI638402</td><td>For suturing the graft</td></tr><tr><td>Black polyamide monofilament suture, 6-0</td><td>AROSurgical Instruments</td><td>SN-1956</td><td>For musculature and skin closure</td></tr><tr><td>Non-woven sponges</td><td>McKesson Corp.</td><td>94442000</td><td></td></tr><tr><td>Absorbable hemostat</td><td>Ethicon</td><td>1961</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>18 G 1.5 in, Needle</td><td>BD</td><td>305190</td><td></td></tr><tr><td>25 G 1 in, Needle</td><td>BD</td><td>305125</td><td></td></tr><tr><td>30 G 1 in, Needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-700</td><td></td></tr><tr><td>Warming pad</td><td>Gaymar</td><td>TP-22G</td><td></td></tr><tr><td>Trimmer</td><td>Wahl</td><td>9854-500</td><td></td></tr></tbody></table>

implantation of inferior vena cava interposition graft in mouse model

Kemik iliği mononükleer hücreler (BMCS) ile ekildi biyobozunur iskelelerinin genellikle doğumsal kalp anomalileri tedavi rekonstrüktif cerrahi için kullanılır. Uzun süreli klinik sonuçları darlık önemli insidans, ancak mükemmel açıklık oranları gösterdi. Vasküler neotissue oluşumu hücresel ve moleküler mekanizmalarının araştırılması ve doku mühendisliği damar grefti (TEVGs) içinde darlık gelişimini önlemek için, biz yaklaşık 1 mm iç çapı ile greft bir fare modeli geliştirdi. İlk olarak, TEVGs bir poliglikolik asit dokunmamış imal edilmiş biyolojik olarak parçalanabilir boru şekilli iskeleler monte edilmiştir ε-kaprolakton ve L-laktid kopolimeri ile kaplanmış ağ hissetti. Iskeleler, daha sonra bir dondurarak kurutucuda yerleştirilen 24 saat boyunca vakumlu ve hücre tohumlama kadar bir desikatör içinde depolanmıştır. İkinci olarak, kemik iliği donör farelerden toplandı ve tek çekirdekli hücreler yoğunluk gradyan santrifüjü ile izole edilmiştir. Üçüncü olarak, yaklaşık bir milyon hücre idibir yapı iskeleti üzerine ekildi ve O / N inkübe Son olarak, tohumlanmış iskeleler sonra C57BL / 6 farelerinde Infrarenal vena kava araya girme greft olarak implante edildi. Implante greftler tromboembolik komplikasyonlar veya anevrizmal oluşum kanıt olmadan mükemmel açıklık (&gt;% 90) gösterdi. Bu fare modeli anlayış bize yardım ve TEVG içinde neotissue oluşumu hücresel ve moleküler mekanizmaları miktarının olacaktır.

TEVG implantasyonu şematik, Şekil 1 &#39;de gösterilmiştir. Kemik iliği, verici bir fare hasat edilmiştir ve mono nükleer hücreler yoğunluk santrifüj ile izole edildi ve daha sonra biyolojik olarak parçalanabilir bir yapı iskeleti üzerine ekildi. Numaralı seribaşı iskeleleri O / N bekletilir ve bir alt vena kava greft interpozisyonu gibi bir alıcı fareye implante edildi. Şekil 2, PGA-P (CL / LA) iskele taramalı elektron mikroskobu göster...

Watch this Scientific Journal Video about Implantation of Inferior Vena Cava Interposition Graft in Mouse Model at JoVE.com

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Hücresel ve moleküler neotissue oluşumu hakkındaki bilgi geliştirmek için, TEVG bir fare modeli yakın zamanda geliştirilmiştir. Greftler C57BL / 6 farelerinde Infrarenal vena kava araya girme greft olarak implante edildi. Bu model bizim klinik araştırmada elde edilenlere benzer sonuçlar elde, ama çok kısaltılmış zaman zarfında.

Fare Modeli Inferior Vena Cava İnterpozisyon Greft İmplantasyonu

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

implantation-of-inferior-vena-cava-interposition-graft-in-mouse-model

Research

JoVE Journal

Medicine

11.8K Görüntüleme.  Nationwide Children's Hospital.  Hücresel ve moleküler neotissue oluşumu hakkındaki bilgi geliştirmek için, TEVG bir fare modeli yakın zamanda geliştirilmiştir. Greftler C57BL / 6 farelerinde Infrarenal vena kava araya girme greft olarak implante edildi. Bu model bizim klinik araştırmada elde edilenlere benzer sonuçlar elde, ama çok kısaltılmış zaman zarfında. 

Video: Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Farelerde Heterotopik Kalp Nakli

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Biyoloji

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Modifiye Manşet Tekniği Sıçangiller Servikal Kalp Nakli Modeli

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Tıp

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Heterotopik Kalp Nakli bir Fare Modeli Kullanarak Pulmoner Vana Nakli

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Farelerde Heterotopik Servikal Kalp Nakli

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Inferior Vena kava stenozu: Derin Ven Trombozu fare modeli

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

İmmünoloji ve Enfeksiyon

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Ven interpozisyonu Model: baypas açıklığının Eğitim için uygun bir model

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Vasküler Manşetler Kullanılarak Fare Heterotopik Servikal Kardiyak Transplantasyonu

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

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 ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Abdominal Fare Kalp Nakli Modelinde Kan Devresi Rekonstrüksiyonu

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Fare Servikal Heterotopik Kalp Transplantasyon Modeli için Modifiye Manşet Tekniği

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...