Roma Wieczorek ve onların mükemmel düzenleme yardım için Peter Kurdybacha ve Leon Decker ve Uli Heuter onların mükemmel teknik destek için teşekkür etmek istiyorum.

hayvan konular içeren yordamlar onaylanan kurumsal hayvan bakım ve kullanım Komitesi (IACUC) tarafından RWTH Aachen Üniversitesi, AZ 84-02.04.2012.A234.
 Not: yordamı farelerde yetişkin erkek yaban tip CD1 arka plan ile kullanma uygulaması gösterilmiştir. Fareler önce ve yemek, özel veteriner kontrol ve tedavi için uygun erişimi temin ameliyattan sonra bir özel laboratuvar biriminde tutmak. Hayvanlar üzerinden aldıysanız dışında bir haftalık iklimlendirme ameliyat gerçekleştirmeden önce izin.
 1. donör hazırlanması <ol><li>steril malzeme ve aletleri steril koşullar enfeksiyonları önlemek için ameliyat sırasında korumak için kullanın.</li>
	<li>Her fare % 1.5 karışımı ile birim isoflurane ve % 100 oksijen bir yüz maskesi ile anestezi. Fare sırtüstü pozisyon ve teyp bir platformda ayakları için ameliyat masasına yattı. Onun refleksleri fare yeterince anestezi emin olmak için arka ayaklarına pinching tarafından kontrol edin. İşlem sırasında kurutma önlemek için göze oftalmik merhem yerleştirin.</li>
	<li>Tüm saç depilatory bir jel kullanarak karından kaldırın veya bir tıraş makinesi kullanın. Steril koşullarda işlemini gerçekleştirin. Scrubs klorheksidin ve steril su alternatif ile karın dezenfekte.</li>
	<li>Makas veya neşter ile donör aort Ensizyon karın üzerinden kaldırın. Bağırsak el ile sağa doğru çekin. Hafifçe el ile bağırsak üzerinde toz ücretsiz eldiven kullanarak yan yansıtır.
	<ol><li>Bağırsak nemli tutmak için serum ıslak gazlı bez bir parça yerleştirin.</li>
		<li>Uzağa--dan çok dikkatli bir künt diseksiyon cımbızla kullanarak çevreleyen doku abdominal aort teşrih.</li>
		<li>Renal arterler ve onun çatallanma vena kava üzerinden arasında aorta segment cımbızla ayırın.</li>
		<li>Bu segmentte çok dikkatli bir şekilde 11-0 monofilament tek dikiş kullanarak tüm küçük dalları güvenli.</li>
		<li>Aort kaldırmadan önce 0.5 ml (mL) tuzlu çözüm inferior vena kava 50 U heparin içeren enjekte.</li>
		<li>Aorta segment kaldırıldıktan sonra exsanguinate donör hayvanın izin.</li>
		<li>Dokuları tam olarak serum fizyolojik ile durulayın ve buz gibi Salin bir kaba hemen transfer.</li>
	</ol></li></ol> 2. Alıcının hazırlanması <ol><li>% 1.5 karışımı ile alıcı hayvan birimi isoflurane ve % 100 oksijen bir yüz maskesi ile anestezi sonra saç kaldırmak ve (Bölüm 1) dezenfekte. Bir neşter ile leğen kemiği için xiphoid gelen bir orta hat kesi yapmak ve karın duvarları geri çek. Gözlerde kuruluk işlem sırasında önlemek için oftalmik merhem yerleştirin.</li>
	<li>Tuzlu çözüm bağırsaklarında ıslatılmış gazlı bez ve çok yavaşça yerinden hayvan için şal &#39; yolunda.</li>
	<li>İnfrarenal aortada ücretsiz renal arterler arasında proksimale teşrih ve cımbız ile klemple çatallanma.</li>
	<li>Bu segmentte çok dikkatli bir şekilde 11-0 monofilament tek dikiş kullanarak tüm küçük dalları güvenli.</li>
	<li>6-0 tek ipek dikiş ile aort proksimal ve distal kısımları kelepçe.</li>
	<li>Kelepçeler arasında orta aort bölmek ve Lümen açık temizlemek için heparinized serum fizyolojik ile kesme biter sulamak.</li>
	<li>Greft aort herhangi bir burulma düzgün donör ve alıcı (yerleştirerek önlemek için dikkat çekici 11-0 monofilament ile dikiş tarafından takip alan gemi eklenen besleme gemi ucuyla orthotopic bulunduğu yer şekil 1) 10.</li> <li>dikkatle bitişik harfler bir izlenebilirliği anastomoz yürütülmesi sonra serbest bırakmak. Distal klempi piyasaya çıkış tarihi. Alçak basınç birlikte proksimal yüksek basınç yan bırakmadan önce duvarlar tutan bu sonuç.</li>
	<li>Greft hemen sıvı ve görünür nabzını kontrol edin. Yavaşça ipek kalıntıları çıkarmak. 1-2 mm en iyi örtüşme uzunluğudur donör ve alıcı aort arasında.</li>
	<li>Karın karın boşluğu döndürmek ve bütün kapatmak ile çalışan 3-0 Poliglikolik asit dikiş yarası.</li>
	<li>Fare buprenorfin vermek (0,05 mg/kg vücut ağırlığı subkutan (SC)) anestezi sonlandırmadan önce.</li>
	<li>Bir hayvan, tamamen bilinçli kadar sahipsiz bırakmayın. Ağrı yönetmek terapi buprenophine 0,05 mg/kg vücut ağırlığı ile yönetilen SC ameliyat sonrası üç gün boyunca günde üç kere Kurumsal Gözetim vücut tarafından onaylanmış.</li>
	Yukarıda açıklanan <li>hasat, doku anestezi için olarak alıcı fareler ve floş gemi fosfat tamponlu tuz çözeltisi (PBS) ile % 4 formaldehit/PBS tarafından takip (pH 7.4 =) kardiyak ponksiyon tarafından. Greftler yavaşça çıkarın. %4 formaldehit/PBS gecede fiksasyonu sonra işlem örnekleri daha fazla ve parafin içinde embed.</li>
</ol>

<ol>
	<li>Rowinska, Z., 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=Non-+invasive+in+vivo+analysis+of+a+murine+aortic+graft+using+high+resolution+ultrasound+microimaging.">Non- invasive in vivo analysis of a murine aortic graft using high resolution ultrasound microimaging.</a> Eur J Radiol. 81 (2), 244-249 (2012).</li><li>Daugherty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+atherosclerosis.">Mouse models of atherosclerosis.</a> Am J Med Sci. 323 (1), 3-10 (2002).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc .Biol. 24 (3), 429-434 (2004).</li><li>Zernecke, A., Shagdarsuren, E., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+atherosclerosis:+an+update.">Chemokines in atherosclerosis: an update.</a> Arterioscler Thromb Vasc Biol. 28 (11), 1897-1908 (2008).</li><li>Chereshnev, I., 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=Mouse+model+of+heterotopic+aortic+arch+transplantation.">Mouse model of heterotopic aortic arch transplantation.</a> J Surg Res. 111 (2), 171-176 (2003).</li><li>Koulack, J., McAlister, V. C., Giacomantonio, C. A., Bitter-Suermann, H., MacDonald, A. S., Lee, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+aortic+transplant+model+of+chronic+rejection.">Development of a mouse aortic transplant model of chronic rejection.</a> Microsurgery. 16 (2), 110-113 (1995).</li><li>Engelbrecht, G., Kahn, D., Duminy, F., Hickman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+rapid+technique+for+renal+transplantation+in+the+rat.">New rapid technique for renal transplantation in the rat.</a> Microsurgery. 13 (6), 340-344 (1992).</li><li>Baxter, K., Hao, P. M., Howden, B. O., Saunder, A., Jablonski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+of+abdominal+heart+transplantation+in+the+rat.">Modified technique of abdominal heart transplantation in the rat.</a> J Heart Lung Transplant. 17 (11), 1057-1064 (1998).</li><li>Dambrin, C., Calise, D., Pieraggi, M. T., Thiers, J. C., Thomsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice:+a+new+model+of+allograft+arteriosclerosis.">Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.</a> J Heart Lung Transplant. 18 (10), 946-951 (1999).</li><li>Siemionow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+microarterial+anastomoses:+end-to-end+versus+end-in-end+(sleeve)+technique.">Histopathology of microarterial anastomoses: end-to-end versus end-in-end (sleeve) technique.</a> J Hand Surg Am. 15, 619-625 (1990).</li><li>Charo, I. F., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354 (6), 610-621 (2006).</li><li>Sun, H., 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=Improved+surgical+technique+for+the+establishment+of+a+murine+model+of+aortic+transplantation.">Improved surgical technique for the establishment of a murine model of aortic transplantation.</a> Microsurgery. 18 (6), 368-371 (1998).</li><li>Guo, L., Agarwal, A., George, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice+for+the+study+of+vascular+disease.">Orthotopic aortic transplantation in mice for the study of vascular disease.</a> J Vis Exp. (69), e4338 (2012).</li></ol>

Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Bu çalışmada önce çeşitli diğer nakli modelleri farelerde iyice analiz3,6,7,10,13,14vardı. O bizim ölçütleriyle eşleşen ve kol anastomoz geleneksel uçtan uca dikiş yöntemleri ile karşılaştırıldığında yüksek bir güvenilirlik gösterdi aort nakli Dambrin vd tarafından değişiklikleri ile kol tekniği kullanarak model seçildi 1 , 10.
Bu önemli ölçüde azaltılmış çapraz klemp süresi ameliyat sırasında aorta zarar en aza ile birçok yönden olumlu bir tekniktir. Tromboz sıklığı düşük gemi kalibre donör ve alıcı arasında bir potansiyel uyumsuzluk kaçınarak ek olarak gözlendi. Yukarıdaki gözlemleri bu teknik son derece aort nakli farelerde vasküler hastalığı araştırma için uygun olun.
Bir takip çalışması bir ultrason nakli sonra 8 hafta gerçekleştirildiği, herhangi bir önemli değişiklik algılanmadı. Bu varsayımlar aort cerrahisi sırasında herhangi bir hasar en az1olacağını doğruladı.
Bu makalede sunulan aşılama yordam hiçbir bozulma greft bütünlüğü ve işlevi hem de güvence altına alır. Bu nedenle, bu deneysel nakli model dejeneratif damar hastalığı genetik farelerde gelecekteki moleküler ve farmakolojik araştırmalar için değerli bir araç olarak hizmet söylenebilir.
İnanıyoruz video rehber bu basit arterio-arter modeli kullanımını gösteren malzeme öğretim olarak işlev görebilir ve daha verimli tartışma vasküler patolojiler birçok önemli konularda katkıda bulunacaktır. Bu çok hızlı anastomoz yöntem genetiği farelerde damar hastalıkları eğitim için kullanılabilir. Bir değişiklik ile ekimi kombine anevrizma modeli olarak da kullanılabilir.
İşlem sırasında kritik noktaları vardır. Dikiş yerleştirilmesi en önemli adım olduğunu. Cerrah tarafından donör ve alıcı doğru hizalaması herhangi bir burulma aort önlemek için dikkat çekmek vardır. Kelepçeler dikkatle anastomoz muayene sonra kaldırılır. Distal klempi her zaman ilk alçak basınç duvarlar birlikte önce proksimal yüksek basınç yan yayın holding sonuçlanan piyasaya sürülmelidir. Düzgün bir şekilde yayın dizisi takip sonucu kanasın.

Bir önceki çalışmada belirttiği gibi büyük ilgi belirli vasküler yanıt greft tarafından neden olduğu ve arteriogenic çevre ile bağlı bazı sistemik faktörler arasında ayrımcılık sağlayan fare aort nakli modelleri için ödenen 1 , 2 , 3. eleme ve transgenik fareler durumu önemli bir rol oynar en büyük etken olduğunu. Böyle bir model onların katılımı belirlenmesi ve ateroskleroz ve anevrizma oluşumu4, gibi dejeneratif damar hastalığı gelişimi ile ilişkili yeni patofizyolojik yolları belirleme imkanı sunuyor 5.
Bu bir iç iskemi/reperfüzyon aşılama sırasında yaralanma nakli için amaçlanan damarları görünebilir dikkati çekiyor. Bu nedenle, greft bütünlüğü veya ameliyat sonrası dönemde beklenmeyen bir enflamatuar reaksiyon ile belirli sorunları oluşumunu muhtemelen dejeneratif vasküler hastalıklar3 patofizyolojik değişiklikler iyileştirmelerden dışarıda bırakılamaz ,4,5,6,7. Kol anastomoz damarları arter anastomoz az bir milimetre çapında için alternatif sıra içinde yöntemidir ve başarılı bir şekilde daha sonra adapte böbrek ve kalp nakli Sıçanlarda aort için uygulanmıştır Dambrin ve ark. tarafından farelerde nakli 8 , 9 , 10 , 11.
Aort hasar kol nakli tekniği kullanılarak bu ortalama sadece 20 dakika süren nedeniyle bir çok düşük teknik başarısızlık oranı ile en aza indirgemek. Önceki sonuçlarımız kol tekniği1kullanarak nakli sonra bir isograft vivo içinde mükemmel fonksiyonel ve yapısal özelliklerini gösterdi. Dambrin ve ark. tarif bu kısa bir öğrenme eğrisi başarı oranı % 78'i şey bitti sonra10. Örneğin8sıçan böbrek nakli kol teknikle trombozu Engelbrecht vd gözlemlemek değil tromboz gibi komplikasyonlar nadirdir.
Fare aort nakli modeli ile kol anastomoses ISO/homogreft reaksiyonlar transplante gemi içinde çalışmak için hızlı ve kolay bir araçtır. Bu video bizim laboratuvarda yapılan aort nakli yordamı gösterir. Bu nakli model vasküler hastalık altta yatan patolojik mekanizmaları tanımlamada yararlı olabilir ve moleküler ve farmakolojik müdahaleler12daha fazla değerlendirilmesi için katkıda bulunabilir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Halsey Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
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		<tr>
			<td>Dumont #5-45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35&#8232;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps -</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Lexer-Baby Scissors</td>
			<td>Fine Science Tools</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>Aesculap, Germany</td>
			<td>Typ OC498R</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Suture Forceps</td>
			<td>Geuder, Germany</td>
			<td>19015</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 silk black (Silk)</td>
			<td>Deknatel, Research Triangle Park NC, USA</td>
			<td>18020-60- FST</td>
			<td></td>
		</tr>
		<tr>
			<td>11-0 monofilament (Ethilon)</td>
			<td>Ethicon, Norderstedt, Germany</td>
			<td>EH7438G</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 polyglycolic acid suture (Serafit)</td>
			<td>Serag-Wiessner, Naila, Germany</td>
			<td>60203214</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
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			<td>0.9% saline</td>
			<td>Any genericon</td>
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			<td>Buprenorphine</td>
			<td>Any genericon</td>
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			<td>Bepanthene eye and nose cream</td>
			<td>Bayer, Germany</td>
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			<td>Microscop</td>
			<td>Zeiss</td>
			<td>Opmi MDO/S5</td>
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			<td>Vaporiser</td>
			<td>Eickemeyer</td>
			<td>TEC3</td>
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			<td>Ultrasound</td>
			<td>Vevo, Canada</td>
			<td>770,2100</td>
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	</tbody>
</table>

using the sleeve technique in a mouse model of aortic transplantation - an instructional video

Orthotopic aort nakli kol tekniği kullanarak yaralanma ile başarısızlık oranı sadece % 10-20 aortaya azaltır. Kısa ve kolay çalışmalar ISO/allo greft izin 20 dk ortalama kol yöntemini kullanan farelerde aort anastomose vakti gelmişti. Aşağıdaki makalede, bizim Laboratuvarda kullanılan aort nakli yordam. Fareler % 1.5 birim isoflurane ve yüz maskesi ile % 100 oksijen karışımı ile anestezi. Bu noktada, renal arter ve onun çatallanma arasında aorta segment vena kava, serbestçe hazırlanan ve clampedat ayrılmış olan tek ipek dikiş proksimal ve distal kesimi. Aort kaldırma önce heparin içeren bir serum fizyolojik çözüm inferior vena kava enjekte ettiler. Sonra aort kelepçeler arasında kesildi ve serum fizyolojik heparin çözüm Lümen temizlemek için kullanılmıştır. Monofilament dikiş kol teknikle abdominal aort orthotopic pozisyonda nakli için kullanıldı.

Yükseltilmiş bir tromboz riski olsa anestezi gözlenen hiçbir fiziksel bozulma ile 15-30 dakika içinde fareler kurtarıldı. Ultrason analiz Postoperatif takip sırasında kullanıldı. Çalışmada kullanılan yaban tipi fareler hiçbir değişiklik onların Lümen boyutta sergiledi. Buna göre ne stenoses ne de aneurysmal oluşumları gözlenmiştir. Organ nakli hayvanlar plak geliştirme (Şekil 2) damar duvarlarının sergilemek başarısız oldu.
İmmunohistology gibi geleneksel boyama prosedürler plak, desen düz kas hücreleri ve makrofajlar birikimi belirlemek için kullanılabilir. Histolojik çalışma ve immunohistokimyasal boyama greft bütünlüğünü sınamak için aşılama sonra 6 hafta gerçekleştirilen. Histolojik boyama (Hematoksilen ve Eozin (o) ve immunohistokimyasal (düz kas aktin (SMA) ve makrofaj (MAC2)) boyama (şekil 3) bize gösterdi değişmeden düz kas hücreleri, sağlam endotel hücre astar ve Hayır dağıtım şekilleri intima hücrelerde birikimi. Bu bulgular Hayır önemli lezyon veya hücre harekete geçirmek aşılı gemiler (şekil 3) algılandığını göstermektedir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54915/54915fig1.jpg"> 
Resim 1 : Kollu tekniği. Abdominal aort kol tekniği kullanarak nakli. Bu yordamda bulaşan aort yüzeysel ısırıkları besleme gemi mevcut ile orthotopic pozisyonda yerleştirilmişti. <a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54915/54915fig2.jpg"> 
Resim 2 : İntraoperatif ve ultrason resimleri. İntraoperatif (nekropsi) görünümü greft 6 hafta sonra nakli(a), üç boyutlu ultrason (B) ve B-modu görünümü (C) örnekleri. Ameliyat sonrası takip ultrason kullanılarak yapılmıştır. Resimleri herhangi bir değişiklik ile greft açıklık Lümen boyutlu olarak göster. Ayrıca, hiçbir stenoses veya aneurysmal oluşumları gözlenmiştir. <a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54915/54915fig3.jpg"> 
Şekil 3 : Histolojik ve immünhistokimya görüntüler. 6 hafta sonra nakli transplante hayvanların histolojik ve immünhistokimya temsilcisi görüntüler. Önemli lezyon yok içinde transplante aortas Histoloji tarafından tespit edilmiştir (Hematoksilen ve Eozin, o, 100 X büyütme, ölçek çubuğu = 50 µm), (düz kas aktin SMA (kırmızı) veya makrofaj MAC2 (yeşil), 200 X büyütme, ölçek çubuğu = boyama immunohistokimyasal 25 µm). Çekirdeklerin DAPI tarafından (mavi) karşı lekeli. <a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video at JoVE.com

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Biz farelerde kol tekniği kullanarak bir orthotopic aort nakli modeli mevcut. Bu damar hastalığı çalışmalarda istihdam bir çok hızlı anastomoz yöntemdir.

Kol tekniği aort transplantasyonu - eğitici bir Video bir fare Model kullanma

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

using-sleeve-technique-mouse-model-aortic-transplantation-an

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.9K Görüntüleme.  Ruhr- University Bochum. Biz farelerde kol tekniği kullanarak bir orthotopic aort nakli modeli mevcut. Bu damar hastalığı çalışmalarda istihdam bir çok hızlı anastomoz yöntemdir.

Video: Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Ortotopik Aort Nakli: Rat Modeli Kronik Vaskülopati Geliştirme Çalışması

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Tıp

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Bir Fare Modeli Utero Organ Nakli

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Damar Hastalığı Çalışmaları Farelerde Ortotopik Aort Nakli

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Rekabetçi bir mürin Kemik İliği Transplantasyon Modelinde Donör Hücre Aşı belirleme Kantitatif Gerçek-zamanlı PCR kullanarak

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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 ...

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Oluşturma ve diyabetik yara iyileşmesi manken bir yağ elde edilen kök hücre (ASC) Sheet nakli

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Fare modeli değiştirilmiş manşet tekniği kullanarak pankreas nakli için

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Modifiye Dikişsiz Manşet Tekniği Kullanılarak Murine Servikal Aort Transplantasyon Modeli

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...