Biz protokol, Dr Michael Simons, Dr Kip Hauch ve genel tartışma için laboratuvarlarında her iki üyelerinin ilk denemeleri için Dr Kelly Stevens teşekkür ederim. Bu çalışma NIH hibe HL087513 ve P01 HL094374 tarafından destek.

1. Başlamadan önce hazırlıklar <ol><li> , Sırasıyla, PBS içerisinde Vazodilatör tampon (4 mg / L Papaverin + 1 gr / L PBS içinde Adenosin) veya 4% paraformaldehit (PFA) ile basınçlı perfüzyon aparatı her bir tarafında doldurun. </li><li> 1:100 Heparin 0.1 ml (5000U/ml hisse senedi) ile doldurma ve yukarı eğim ile ~ 120 derecelik bir açıyla iğne bükerek bir 1/2cc İnsülin Şırınga (bir srekli 29G ½ &quot;iğne ile) hazırlayın. Mı 0.3 ml doymuş KCl çözeltisi ile doldurulmuş 1 ml şırınga (26G bir ½ &quot;iğne ile) ile aynı. </li></ol> 2. Kalp teşhir ve aort cannulating <ol><li> Seçtiğiniz anestezik kullanarak fare uyutmak. (Biz bir Ketamin / Ksilazin karışımı aşırı dozda kullanın:. 130 mg / kg Ketamin ve tuzlu su içinde 8.8 mg / kg Xylazine IP enjeksiyon) </li><li> Anatomi tepsisi, ventral tarafı yukarı üzerine anestezi fare Pin. Orta hat kesi ile karın boşluğu açın ve cildi geriorganları açığa. Posterior Vena Kava (PVC) bir bölge ortaya çıkarmak bir yana bağırsak taşıyın. </li><li> PVC içine heparin solüsyonu enjekte edilir. Eğer iğne ayıklamak gibi, sızıntı önlemek ve PVC duvar pıhtılaşması ve mühürler kadar birkaç saniye tutun için bir pamuk uçlu aplikatör ile iğne deliği kapsar. Fare dolaşımı boyunca dağıtmak için Heparin için 2-3 dakika bekleyin. </li><li> Eğer kalpte gözlemlemek, böylece diyafram ve göğüs kafesini parçalara ayır. Yavaş yavaş kalp tutuklamalar kadar PVC KCl çözeltisi enjekte edilir. </li><li> Diyaframın bölgenin anterior zedelemeden, diyafram ve tüketim fare posterior bölümünün altındaki bütün organları çıkarın. Kalp proksimal kısmı sonraki adımlarda bulmak kolay yani diyafram yakınında PVC kesmek için dikkatli olmanın, diyafram çıkarın. </li><li> Aort kesik ucuna bulun. Aort altında anterior birkaç milimetre sağa sola 6-0 örgü ipek sütür biri uzun boyu yerleştirinm sütür geri kendisini iki katına olduğu gibi kesik uç. Aort altında sütür 2 adet vardır bu nedenle yarısında bu uzun sütür kesin. Aorta (Şekil 1A, B) kesik ucunu angiocatheter yerleştirin ve yerine angiocatheter tutun ve dışarı sızmasını aort içinde herhangi bir geri basıncı önlemek için bir çift düğüm, her dikiş kravat. </li></ol> 3. Perfüzyon ve Microfil enjeksiyon <ol><li> Basıncı perfüzyon aparatı (Şekil 2) angiocatheter bağlayın ve 100-110 mmHg bir sürüş basıncına perfüzyon cihazı pompalayarak vazodilatör tamponu (Şekil 1C) gemiler perfüze başlar. Tampon sıvı sağlayarak koroner arterler ile perfüze olup olmadığını kontrol edin çift PVC çıkarken. En az 3 dakika serpmek devam veya PVC çıkan sıvı netleşene kadar. (Perfüze ise sonraki adımlar ile devam edin.) </li><li> Kaburga ve pin geri (veya kaldırmak) kalp duyurmak için göğüs kafesi parçalara ayır. Bir kere maruz kalan, caKalp gazlı bez tampon bulanmış parça kalbimin üstüne tampon damla sıkarak kuruması için değil reful. Aort arkı maruz timus uzak temizleyin. Sıvı sağlamak için 6-0 örgü ipek sütür kullanılarak üç büyük aort dalları Ligate yerine bu büyük, düşük dirençli damarlar (Şekil 1D) aracılığıyla daha koroner arterler üzerinden saptırılır. </li><li> 15 dakika boyunca sabitleştirici ile kalp serpmek, ardından en az 2 dakika süreyle Vazodilatasyon tamponu ile durulanır. Bu arada, enjeksiyon sonrası kalp (1E) dışarı sızmasını Microfil önlemek için hem ön Vena Cavae ligate. Yeri PVC ve aort etrafında dikişler ama doldurduktan sonra kadar onları sıkmayın. </li><li> Microfil hazırlayın (as reaktiflerin tabloda belirtildiği gibi) ve 1 ml şırınganın içine yerleştirin. Kateter (Microfil şırıngaya perfüzyon boru geçerken hava kabarcıklarının girişini önlemek amacıyla) kaplayacak kadar su ile diseksiyon tepsi doldurun. Perfüzyon ap ayırınkateter paratus ve hazırlanmış Microfil şırınga bağlanır. </li><li> Koroner arterlerin iyi dolgu (Şekil 1F, 3A) kanıtlanıncaya kadar aorta Microfil enjekte: arterler ilk dolduracak ve sonra Microfil Microfil rengi ile doku basması gibi kılcal damarlar içine &quot;dökülebilir&quot; olacaktır. Bir kez venöz tarafında, Microfil hidrofobik yapısı nedeniyle küçük gemilere çıkar başlangıçta bağımsız bir küre gibi görünmeye neden olur. Sürekli bir sütun damarları doldurur kadar Microfil enjekte devam edin. Microfil damarlar içinde sürekli olduğunda komple dolum belirgin olacak ve PVC ile çıkarken. </li><li> Dolum işlemi tamamlandıktan sonra, hızlı bir şekilde daha önceden damarlarının dışarı Microfil sıkma gelen kardiyak doku elastik doğası önlemek için PVC ve aorta etrafında yerleştirildi dikişlerle sıkmak. </li><li> (Diseksiyon tepsiden su ile ıslatılmış ıslak gazlı bez ile kalp kapak) Onun kurumasını önlemek ve Microfil polimerize kadar oda sıcaklığında yaklaşık 1 saat bekletin için. Böyle kaldırırken veya kalp arkasında dolu gemilerin erken bir görünüm elde etmek için bir girişim kalp dönüm olarak polimerizasyon işlemi esnasında kalp üzerinde herhangi bir dış baskı, kaçının. Bu Microfil tatili neden kalp bir daha elastik bir alana bazı gemilerin Microfil sıkmak olabilir. </li><li> Kalp çıkarın ve 4 gece 4% PFA post it-fix ° C Daha sonra 4 ° C&#39;de% 70 etanol içinde depolamak Kalp damar artık μCT görüntüleme için hazırdır. </li></ol> 4. Temsilcisi Sonuçlar Etkili Microfil ile perfüze olan gemiler gemi boyunca sürekli, kesintisiz Microfil (Şekil 3A) olacaktır. Koroner damarlarda dolum kapsamı göz tarafından değerlendirilecek olabilir; damarlar epicardially 18 bulunur ve kolayca (Şekil 3A, ok) görülebilir; arterler,hangi 18 daha intramiyokardiyal olan kalbin yüzeyine (Şekil 3A, ok) ile de görülebilir. Kardiyak doku kılcal damarların çok yüksek bir yoğunluğa sahip olarak kapiller dolum, de bellidir, ve bu nedenle, ne zaman kılcal dolgu, kardiyak doku Microfil rengini (Şekil 3A, yıldız) ile floş olacaktır. Böylece, doldurmak için başarısız herhangi bir damar ağları Microfil eksikliği (Şekil 3B, C) bağlı fark olacaktır. Microfil hidrofobik doğanın kendi içine kasılmasına ve dolu gemi içinde &quot;sonları&quot; neden neden çünkü Microfil (Şekil 3B&#39;de yıldız) süreksizlikler sık ​​görünür. Damarları içindeki basıncı kalpten damar çıkış noktalarının tie-off&#39;lara uygun yoluyla sürdürüldüğü takdirde, bu &quot;molaları&quot; azaltılabilir. Diğer süreksizliklerin mikrofiltre içinde hava kabarcıkları neden olabilir. Perfüzyon ap geçerken hava girişini önlemek için, angiocatheter tamamen su altında olduğundan emin olun<html> Microfil şırıngaya paratus. Bir hava kabarcığı tanışınca, genellikle balonu koroner damarların üzerinden itti ve dışarı dek Microfil perfüzyon devam ederek basitçe kaldırılabilir. Vasküler yataklı bir bölümünü (Şekil 3B, ok) bloke halinde vasküler ağlar tamamen dolgu olmayabilir. Heparin kan pıhtısı oluşumunu inhibe ederken, zaman zaman tıkanmalara hala prosedür öncesi veya başka bir bilinmeyen faktörler nedeniyle başlayan eksik heparin perfüzyon nedeniyle oluşabilir. Bir tıkanma meydana gelirse, vasküler dolgu tamamlamak için blokaj yerinden oynatmamaya için hiçbir yöntem, bildiğimiz kadarıyla yok. Çok az basınç Microfil tüm vasküler yatak ve kılcal ağlar (Şekil 3C) zorla olmayacak gibi, dolum esnasında kullanıldığı takdirde eksik bilgi girilmesi de neden olabilir. Tersine, çok fazla basınç kılcal (Şekil 3D) çevredeki dokuya Microfil patlaması ve extravasate neden olabilir. files/ftp_upload/3740/3740fig1.jpg &quot;alt =&quot; Şekil 1 &quot;/&gt; Şekil 1.. Microfil perfüzyon düzeninin bakış. (A) aort ve PVC diyaframın yaklaşık seviyesinde kesilir. (B) aort bir angiocatheter ile kanüle edilir. (D) arkus kapalı üç ana kolu bağlandı iken (C) Vazodilatasyon tampon baskı perfüzyon aparatı (Resim mevcut değil) tarafından tahrik olan gemiler, içinden perfüze edilmiştir. Her iki ön Vena Cavas ligate edilir ise (E) 4% PFA koroner içinden perfüze edilmiştir. Bu PVC&#39;den çıkmadan gözlenene kadar (F) bir şırınga kullanılarak, Microfil koroner içinden perfüze edilmiştir. <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="Şekil 2."/> Şekil 2. Perfüzyon Aparatı. İki Erlenmeyer matara, Vazodilatasyon tampon veya% 4 PFA ya dolu, her biri katıldı ve sidearms bağlı borular aracılığıyla tahrik edilmektedir. Sistemin bir pompa aracılığıyla elle basınçlı haletermometre ve bir basınç ölçer izleme ve basınç bakım izin vermek için şişeler birine bağlanır. Küçük tüpler kauçuk tıpa ile ve aşağı her şişede sıvı içine uzatmak. Basınç bu küçük tüpler dışarı her şişesi sidearms pompalar sıvı giren. Tüpler sonra sadece bir kerede bir şişesi akmaya sıvı sağlayan bir musluk de birleştirebilirsiniz. <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="Şekil 3,"/> Şekil 3. Microfilled kalpleri tadın. (A) de dolu gemiler (varsa) Microfil tatili kaç olacak ve kalp dokusu dolu kılcal (yıldız, ve C ile karşılaştırmak) nedeniyle Microfil ile tinged rengi olacak. Her iki arter (ok - Sol ön inen arter) ve damarlar (ok - Sol Koroner Damar) kalp yüzey boyunca görülebilir. (B) Tam Mikrofon önlenir bazı damarlarda Microfil in sonlarının (yıldız işareti) yanı sıra, tıkanmalar olan bir kalppenetrasyon rofil. Bloke damarlar kan perfüzyon işlemi sırasında dışarı atılır değildi olarak, kırmızı (ok) kalır. Tam olarak dolduruldu damarları ile (C) bir kalp. Doku Microfil kılcal damarları içine nüfuz etmedi belirten Microfil ve sarı renk üzerine almadığını dikkat edin. (D) Bir kalp burada Microfil çevreleyen doku (ok) içine sızmaya neden dolum esnasında kılcal damarların patlaması.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+perfusion+imaging+with+contrast+echocardiography.">Myocardial perfusion imaging with contrast echocardiography.</a> Curr. Cardiol. Rep. 10, 233-239 (2008).</li><li>Leong-Poi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Endogenous+and+Therapeutic+Arteriogenesis+by+Contrast+Ultrasound+Molecular+Imaging+of+Integrin+Expression.">Assessment of Endogenous and Therapeutic Arteriogenesis by Contrast Ultrasound Molecular Imaging of Integrin Expression.</a> Circulation. 111, 3248-3254 (2005).</li><li>Villanueva, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+Targeted+to+Intercellular+Adhesion+Molecule-1+Bind+to+Activated+Coronary+Artery+Endothelial+Cells.">Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells.</a> Circulation. 98, 1-5 (1998).</li><li>Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Myocardial+Blood+Flow+With+Ultrasound-Induced+Destruction+of+Microbubbles+Administered+as+a+Constant+Venous+Infusion.">Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion.</a> Circulation. 97, 473-483 (1998).</li><li>Beckmann, N., Stirnimann, R., Bochelen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Magnetic+Resonance+Angiography+of+the+Mouse+Brain:+Application+to+Murine+Focal+Cerebral+Ischemia+Models.">High-Resolution Magnetic Resonance Angiography of the Mouse Brain: Application to Murine Focal Cerebral Ischemia Models.</a> Journal of Magnetic Resonance. 140, 442-450 (1999).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+MR+angiography+of+intratumoral+vasculature+using+a+novel+macromolecular+MR+contrast+agent.">3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent.</a> Magnetic Resonance in Medicine. 46, 579-585 (2001).</li><li>Nezafat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1-insensitive+T2+preparation+for+improved+coronary+magnetic+resonance+angiography+at+3+T.">B1-insensitive T2 preparation for improved coronary magnetic resonance angiography at 3 T.</a> Magn. Reson. Med. 55, 858-864 (2006).</li><li>Wagner, S., Helisch, A., Ziegelhoeffer, T., Bachmann, G., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+angiography+of+collateral+vessels+in+a+murine+femoral+artery+ligation+model.">Magnetic resonance angiography of collateral vessels in a murine femoral artery ligation model.</a> NMR in Biomedicine. 17, 21-27 (2004).</li><li>Cochet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+angiography+and+velocity+measurement+in+mice+coronary+arteries+at+9.4+T:+assessment+of+coronary+flow+velocity+reserve.">In vivo MR angiography and velocity measurement in mice coronary arteries at 9.4 T: assessment of coronary flow velocity reserve.</a> Radiology. , 254-441 (2010).</li></ol>

Fareler, Washington Üniversitesi Kurumsal Hayvan Bakımı ve Kullanımı Komitesi tarafından ve Sağlık ABD Ulusal Sağlık Enstitüleri (NIH Yayın No 85-23 tarafından yayınlanan Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu göre onaylanmış yöntemlerle ele alındı ) 1996 revize edilmiştir.

Kardiyak doku çok yüksek metabolik talep vardır ve bu nedenle, koroner damar tarafından sağlanan kandan besin ve oksijen sabit bir besleme gerektirir. Damar darlığı ve tıkanma nedeniyle koroner fonksiyonu azaltmak koroner damarlarında, hastalıkları, doku hipoksisi ve iskemiye yol, myokard infarktüsü ve kalp kası onarılamaz hasar riski etkilenen hastalar koyabilirsiniz. Bu gemilerin hastalıklı devlet daha iyi anlaşılması gereklidir ve koroner damarların çalışma yeteneğimizi kritik damarların g?...

<html><table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> 1 ml şırınga </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309.602 </td><td></td></tr><tr><td> Srekli 29G ½ &#39;iğne ile 1/2cc insülin şırıngaları </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309.306 </td><td></td></tr><tr><td> 2 &quot;x 2&quot; tül pedleri </td><td> Med101store.com </td><td> SKU 2208 </td><td></td></tr><tr><td> 24G ¾ &quot;Angiocath IV kateter </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-381.112 </td><td></td></tr><tr><td> 26G ½ &quot;insülin iğnesi </td><td>sıska &quot;&gt; Becton Dickinson </td><td> BD-305.111 </td><td></td></tr><tr><td> Adenozin </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> A9251 </td><td> 1g / Vazodilatasyon Tampon PBS L (Papaverin ile) </td></tr><tr><td> Açılı Graefe Forseps </td><td> <a href="http://www.finescience.com/" target="_blank">Güzel Bilim Araçları</a> </td><td> 11052-10 </td><td></td></tr><tr><td> Pamuk uçlu aplikatörler: 6 &quot;non-steril </td><td> <a href="http://www.cardinal.com/" target="_blank">Cardinal Health</a> </td><td> C15055-006 </td><td></td></tr><tr><td> Kavisli Cerrahi Makas </td><td> <a href="http://www.finescience.com/" target="_blank">Güzel Bilim Araçları</a> </td><td> 14085-09 </td><td></td></tr><tr><td> Kesme stereoskop ve ışık kaynağı </td><td> <a href="http://www.nikon.com/" target="_blank">Nikon</a> </td><td> NA </td><td> NA </td></tr><tr><td> Kesme Tepsi, 11,5 x 7,5 inç </td><td> <a href="http://www.coleparmer.com/" target="_blank">Cole-Parmer</a> </td><td> YO-10.915-12 </td><td> Fareyi aşağı iğnelemeyi için tar ile Dolu </td></tr><tr><td> Güzel Kavisli Forseps </td><td> <a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a> </td><td> FD281R </td><td> Gereken iki </td></tr><tr><td> Heparin, 5000 U / ml stok </td><td> <a href="http://www.apppharma.com/" target="_blank">APP İlaç LLC</a> </td><td> NDC 63323-047-10 </td><td> Su 1:100 </td></tr><tr><td> KCl </td><td> <a href="http://www.fishersci.com/" target="_blank">Balıkçı</a> </td><td> P217 </td><td> H2O Doymuş çözelti </td></tr><tr><td> Ketamin (Ketaset), 100 mg / ml stok </td><td> Fort Dodge, Overland Park, KS, ABD </td><td> NDC 0856-2013-01 </td><td> % 0.9 &#39;s Xylazine ile 130 mg / kg vücut ağırlığı olarak Karışıkdizilmek </td></tr><tr><td> Microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">Akış Tech</a> </td><td> MV-122 (sarı). Diğer renk seçenekleri de mevcuttur. </td><td> Sertleştirici hacim itibariyle% 10 ile, 1:1 karıştırın. Iyi karışık olduğundan emin olmak için sadece enjeksiyon ve vorteks önce hazırlayın </td></tr><tr><td> Steril olmayan Dikiş: 6-0, örgü ipek </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">Harvard Apparatus</a> </td><td> 723287 </td><td></td></tr><tr><td> Papaverin </td><td> <a href="http://www.americanregent.com/" target="_blank">Amerikan Regent A.Ş.</a> </td><td> NDC 0517-4010-01 </td><td> 4 mg / Vazodilatasyon Tampon PBS L (Adenozin ile) </td></tr><tr><td> Paraformaldehyde </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> P6148 </td><td> 4% solüsyon olarak hazırlandı </td></tr><tr><td> Perfüzyon Aparatı </td><td></td><td></td><td> Bakınız, şekil 2 </td></tr><tr><td> Bahar Makas </td><td> <a href="http://www.finescience.com/" target="_blank">Güzel Bilim Araçları</a> </td><td> 15018-10 </td><td></td></tr><tr><td> Xylazine (Anased), 20 mg / gl stok </td><td> <a href="http://www.lloydlab.com/" target="_blank">Lloyd Labs</a> </td><td> ADA # 139-236 </td><td> % 0.9 tuzlu su içinde Ketamin ile 8,8 mg / kg vücut ağırlığı gibi karıştırılabilir </td></tr></tbody></table>

retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

Damarların görüntülenmesi birçok hastalıktan anlamak için giderek önem kazanmaktadır. Çeşitli teknikler görüntüleme damar için mevcut olmakla birlikte, birkaç küçük gemilere 1,2 içeren bir kararın uzanan bir bütün olarak vasküler ağ görselleştirmek edebiliyoruz. Ayrıca, birçok vasküler döküm teknikleri örnek 3-5 daha fazla analiz önlenmesi, çevre doku yok. Bu konularda circumvents Bir yöntem mikro-Bilgisayarlı Tomografi (μCT) &#39;dir. μCT görüntüleme, kararlar &lt;10 mikron tarama damar ağının 3D rekonstrüksiyon üretme yeteneğine sahip olduğunu ve (örneğin, histoloji ve morfometri) sonraki analiz için sağlam 6-11 doku bırakır olabilir. Bununla birlikte, ex vivo μCT yöntemlerle görüntüleme damarlarının damarlarının radyoopak bileşiği ile doldurulmuş olması gerekir. Bunun gibi, μCT görüntüleme tarafından üretilen damarların doğru temsili üzerine olurludurgemilerin güvenilir ve tam dolum. Bu protokol, biz μCT görüntüleme için hazırlık fare koroner damarları doldurmak için bir teknik açıklar. İki hakim teknikler koroner damar doldurmak için var: Bir Langendorff perfüzyon sistemi 15-17 ile kanülasyon ve retrograd aort perfüzyon (veya arkus kapalı bir şube) 12-14 veya ex vivo ile in vivo. Burada özellikle tüm damarlarda dolum sağlamak için tasarlanmıştır vivo aort kanülasyon yöntemi bir tarif. Bütün kılcal gibi vasküler ağın, arter ve ven her iki tarafında, doldurmak için küçük damarlarda serpmek için Microfil denilen düşük viskoziteli radyopak bileşiğin kullanımı. Kaplar basınçlı bir perfüzyon sistemi kullanılarak tamponu ile perfüze ve sonra Microfil ile doldurulur. Bu Microfil küçük yüksek direnç damarları doldurur sağlamak için, büyük dalları emanatin ligatekoroner arterler içine Microfil yönlendirir aorta, gelen g. Bir kez dolum bazı gemilerin Microfil silip atacak gelen kalp dokusunun elastik yapısı önlemek için, tam, biz hemen doldurduktan sonra erişilebilir büyük damar çıkış noktaları ligate. Bu nedenle, bizim teknik komple koroner damar ağı görselleştirme sağlayarak, dolgu ajanı tam dolum ve maksimum kalıcılık için optimize edilmiştir - arterlerin, kılcal damarları ve damarların hem.

Watch this Scientific Journal Video about Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging at JoVE.com

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Koroner damarların görüntülenmesi kardiyovasküler hastalıkların bizim anlayışın geliştirilmesi için çok önemlidir. Burada mikro-Bilgisayarlı Tomografi (μCT) görüntüleme için hazırlık, bir radyoopak silikon kauçuk (Microfil) ile sıçan koroner damar perfüze için bir yöntem açıklanmaktadır.

Mikro Bilgisayarlı Tomografi Görüntüleme için hazırlık olarak retrograd perfüzyon ve Fare Koroner damarları doldurulması

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

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Research

JoVE Journal

Medicine

32.3K Görüntüleme.  University of Washington. Koroner damarların görüntülenmesi kardiyovasküler hastalıkların bizim anlayışın geliştirilmesi için çok önemlidir. Burada mikro-Bilgisayarlı Tomografi (μCT) görüntüleme için hazırlık, bir radyoopak silikon kauçuk (Microfil) ile sıçan koroner damar perfüze için bir yöntem açıklanmaktadır.

Video: Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Segmentasyon ve Mürin Obezite Modelleri Yağ Hacimleri ölçümü X-ray Bilgisayarlı Tomografi Kullanımı

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Tıp

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

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

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

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Mikro-Bilgisayarlı Tomografi ile Çıplak Fareler içine İnsan Yağ Enjeksiyonu Canlılığına Değerlendirilmesi

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Tümör Gelişim Değerlendirme Mikro bilgisayarlı Tomografi ile ve Takibi Tedavi Yanıtın Akciğer Kanseri Fare Modeli

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Doğrudan Serebral emboli görselleştirme için Kombine Yakın kızılötesi Floresan Görüntüleme ve Mikro bilgisayarlı Tomografi

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

Koroner arter kalsifikasyon tespit sigara geçitli hesaplanan tomografi inceden inceye gözden geçirmek

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

Küçük hayvan Pozitron Emisyon Tomografi/Bilgisayarlı tomografi sürekli kan örnekleme arteriyel giriş fonksiyon ölçümü sağlar

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Kronik Hastalığı Olan Büyük Memeli Kardiyak Modellerinin Kontrastlı Mikro Bilgisayarlı Tomografi Görüntülemesinde Doku Hazırlama Teknikleri

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

DUCT: Çift Reçine Dökümü ve ardından 3D Karaciğer Analizi için Mikro Bilgisayarlı Tomografi

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Murine Hind-limb Damarlarının Bilgisayarlı Tomografi Tabanlı Üç Boyutlu Görselleştirilmesi için Örnek Hazırlık

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...