Yazarlar editoryal yardım için, mikro-CT cerrahi yardım için görüntüleme, Edward TH Yeh, MD, ve Rebecca Bartow, Doktora ile yaptığı teknik yardım için MD Anderson Küçük Hayvan Görüntüleme Tesisi Keith Michel teşekkür etmek istiyorum. Bu çalışma, Amerikan Kalp Birliği tarafından kısmen desteklenmiştir.

1. Astar Ozmotik Pompa <ol><li> Pompalar hazırlarken steril teknikler (örneğin eldiven, laminer akış kaputu) kullanın. Ambalajından pompa ve akış moderatör çıkarın. Boş pompa tartmak ve ağırlığı (mg) kaydedin. </li><li> Küçük bir 1 cc şırınga ve künt uçlu 27G dolum tüpü kullanarak, hava kabarcıkları oluşturarak önlemek için, yavaş yavaş pompa doldurun. Çözelti pompanın üst ulaştığında, dolgu dur. </li><li> Aşırı çözüm silin ve dolu pompa tartın. En sulu çözeltiler için, mg olarak ağırlık ul hacmi ile aynıdır. Boş karşı dolu ağırlık farkı, belirtilen ortalama dolum hacminin% 90&#39;dan fazla olmalıdır. Değilse, şırınga ve dolum ile tahliye pompası. , Teknik zorluk yaşarsanız lütfen http://www.alzet.com/products/guide_to_use/filling.html de pompa talimatlarının sorun giderme bölümüne bakın </li><li> Akış moderat gelen beyaz flanş kırmak için forseps bir çift kullanınveya; tüp viraj veya ezmek için dikkatli olun. 3-4 mm hakkında kapsayan, moderatör ya da sonuna kadar bir P50 kateter takın. Kateter ve ekli moderatör girip kateterin distal kısmına takılı şırınga bırakmak için bir şırınga kullanın. </li><li> Kateter pompa yüzeyi ile aynı hizaya gelene kadar pompa içine akış moderatör yerleştirin. Daha sonra, kateterin uzak ucundan şırınga çıkarın. Birinci ila 6 saat, en az (tercihen O / N), fare içine yerleştirilmeden önce pompası için bir 37 ° C inkübatör içinde bir steril% 0.9 tuzlu su çözeltisi içinde veya PBS pompa yerleştirin. Pompa doğum oranı model numarası (örneğin Model 1002 = 14 gün için 0.25 ul / saat). Göre değişir </li></ol> 2. Fare tek taraflı Hindlimb İskemi oluşturma <ol><li> 0.4 L / dk &#39;lık bir akış hızında% 100 O 2% 2-4 arasında izofluran grubu ile fareler anestezi ve depilato ile bacak ve arka bölgede (tercihen ameliyat öncesi gün) tüylerdenry krem. </li><li> Ameliyat öncesi tüm cerrahi aletler sterilize edin. Indüksiyon anestezi odasında fare yerleştirin. Bölüm 2.1 &#39;de açıklanan aynı anestezi ayarları kullanın. </li><li> Fare dışında uyaranlara olmayan duyarlı ve daha sonra izofluran sürekli bir akış (1.5-2.0%) sunan monte burun konisi ile işletim masaya yüzüstü pozisyonda yerleştirin kadar bekleyin. </li><li> Kesi yerinde, anestezik önerilen doz (0.25% (&lt;1 ml / kg) Bupivakain); enjekte% 70 etanol ardından povidon-iyot solüsyonu ile cilt tedavi. Arka (omurganın kenarındaki, scapulaların bölgede) bir 4-6 mm kesi yapmak. Ince forseps ile deri altı membran ayırın, sonra yatar pozisyonda fare açmak% 70 etanol takip ve medial uyluk doğru diz üstü mikro-cerrahi makas ile 0,8-1 cm kesi yapmak bir povidon-iyot solüsyonu ile cilt tedavi, femoral arter yolunu izleyerek. </li><li> Yukarı tünelarkasındaki kesi medial uyluk küçük kılıf boru (6-Fransız) ile gövde yan. </li><li> Yüzüstü pozisyonda fare yerleştirin. PBS çözeltisi ile astarlanmış ozmotik pompa / kateter çıkarın ve uyluk için kılıfın tüp aracılığıyla, kateter kılavuz. </li><li> Içeri pompa yerleştirin ve 6-0 prolen sütür (% 70 etanol ardından povidon-iyot solüsyonu ile cilt tedavi) ile arka kesi kapatın. Yatar pozisyonda fare geri yerlerde, uyluk kesiden maruz kateter bırakarak, kılıf boru çıkarın. Ameliyat bandı ile bacak elde edin. </li><li> Damar ortaya çıkarmak için deri altı yağ dokusu ayırın. Daha iyi damar görselleştirmek için, kesi açmak için bir retraktörü kullanın. </li><li> Vasküler paket erişmek için ince sivri forseps kullanarak hafifçe Pierce membranöz femoral kılıf. Incelemek ve kasık yakın femoral ven ve sinir femoral arter ayırmak, neden özen eskiaşır kanama. </li><li> Iki 8-0 prolen sütür bitişik femoral arter femur bifurkasyon (profunda femoris ve epigastrikan arterlerin kökeni) proksimalinde 2-3 mm kesmek için. </li><li> Uygun uzunlukta kateter kesin ve maddenin hedeflenen teslim sağlamak için yüzeysel fasya bir 6-0 prolen sütür ile güvence, uyluğun anterolateral bölgeye ucu yerleştirin. 6-0 çalışan sütür ile bacak kesi kapatın. Analjezik son bir deri altı enjeksiyonu (buprenorfin, 0.1 mg / kg) yönetmek,% 70 etanol ile izlenen bir povidon-iyot çözeltisi ile cilt tedavi ve kafesine geri fare. Kurtarma sırasında bir ısıtma lambası kullanın. </li><li> Bir 30 dakika geri kazanım süresinden sonra, bacak iskemisi oluşturulması teyit etmek için lazer Doppler görüntüleme için fare hazırlamak. </li><li> Günde iki kez ameliyat sonrası 5 gün için hayvan izleyin. Hayvanın hareketliliği ağrı analiz etmek görülecektir, kafeste atık bir indic olarak görev yapacakyeme ator. Hayvan ağrı veya rahatsızlık yaşanıyor Ayrıca, monitör dönemde belirlemek ve günde iki kez Buprenorfin (0.1mg/kg) bir deri altı enjeksiyon vermek. </li></ol> 3. Görüntüleme Numune Hazırlama <ol><li> Bölüm 2.1 &#39;de belirtildiği gibi fare anestezisi. Yatar pozisyonda ameliyat masasına üzerine fare aktarın. Subkutan damar pıhtılaşma önlemek için heparin 1000 IU enjekte edilir. Fare roket ucu konisi takılı ise, göğüs boşluğu ve alt karın ortaya çıkarmak için yeterince büyük bir kesi yapmak. Infrarenal abdominal aort (IAA) ortaya çıkarmak için hayvanın sol tarafına karın içi organların hareket ettirin. Alt kava damardan aort dikkatle ayırın. </li><li> Kateter yerleştirme sırasında kanamayı azaltmak için proksimal IAA Arter. Daha sonra, IAA küçük bir kesi yapmak. IAA içine ½ G künt bir iğne (bir P10 kateter bağlı) bir 27 takın ve 6-0 sütür ile sabitleyin. </li><li> Dolgu ikiHeparinize tuzlu su ve% 10 nötr ile 20cc şırınga (NBF) tamponlu formalin. Bir kontrast radyoopak silikon kauçuk bileşik ile bir 5cc şırınga doldurun. PD-seyreltici: 1:1.25:0.5 çalışma oranlarında PD-sertleştirici madde PD-bileşiğinin üç maddeler karıştırın. Ajan polimerize önce karışımı (Microfil) sadece 20 dakikalık bir çalışma süresi olduğundan, damar içine enjeksiyon hemen önce kadar MV-sertleştirici eklemek için tavsiye edilir. </li><li> Sağ atrium bir kesim yapmak ve ötenazi ikna etmek ve aşırı sıvı drenajı sağlamak için izofluran aşırı dozda yönetmek. Iğne ucu için tuzlu dolu şırınga takın ve 5 dakika boyunca damar temizlemek için başlar. Tüm kan damarlarının dışarı kızarmış olduğundan emin olun, izofluran kapatın ve yıkama başladıktan sonra roket ucu konisi çıkarın. Daha sonra, yukarıda ayrıntılı olarak kateter (3.2) ve ilave 5 dakika boyunca basıncı serpmek için formalin şırınga takın. Son olarak, kontrast, son şırınga serpmek5 dakika boyunca yavaş yavaş fare içine bir madde. </li><li> İsteğe bağlı: Fotoğraf bir kamera ile damar Microfil sadece arter yapısı perfüze emin olmak için. </li><li> 4% 10 NBF ve mağaza ° CO / kontrast madde polimerizasyon N dolu bir kap içinde fare yerleştirin. 24 saat sonra, diseksiyon araçlarını kullanarak gövde alt yarısı (alt abdominal aort ligasyonu distalinde) üzerindeki deri kaldırmak% 10 NBF yenilemek ve oda sıcaklığında 4 gün daha kaba geri fare yerleştirin. </li><li> Kontralateral ve iskemik arka ayakların ayır ve kemik dekalsifikasyon kolaylaştırmak için, 48 saat boyunca bir formik asit solüsyonu (Cal-Ex II) &#39;de örnek yerleştirin. Daha sonra, 1 saat ve 10% NBF dönmek için akan suyun altında örneklerin yıkayın ve Faxitron (x-ray sistemi kemik dekalsifiye sağlamak için kullanılan) ve mikro-BT görüntüleme ile devam edin. </li></ol>

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Med. 9, 713-725 (2003).</li><li>Hirsch, A. 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=ACC/AHA+Guidelines+for+the+Management+of+Patients+with+Peripheral+Arterial+Disease+(lower+extremity,+renal,+mesenteric,+and+abdominal+aortic):+a+collaborative+report+from+the+American+Associations+for+Vascular+Surgery/Society+for+Vascular+Surgery,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+for+Vascular+Medicine+and+Biology,+Society+of+Interventional+Radiology,+and+the+ACC/AHA+Task+Force+on+Practice+Guidelines+(writing+committee+to+develop+guidelines+for+the+management+of+patients+with+peripheral+arterial+disease)--summary+of+recommendations.">ACC/AHA Guidelines for the Management of Patients with Peripheral Arterial Disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (writing committee to develop guidelines for the management of patients with peripheral arterial disease)--summary of recommendations.</a> J. 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Yazarlar herhangi bir çıkar çatışması beyan ederim. Tüm hayvan prosedürleri Laboratuvar Hayvanları Bakım ve Kullanım Kuralları uyarınca yapılmıştır ve Houston Texas Sağlık Bilimleri Merkezi Üniversitesi&#39;nde Kurumsal Hayvan Bakım ve Kullanım Kurulu (IACUC) tarafından onaylanmıştır.

Burada bacak iskemisi, bir fare modelinde ozmotik ilaç / maddenin dağıtımı için bir yöntem sunulmaktadır. Buna ek olarak, biz damar ağının analizi için 3 boyutlu bir yeniden üretmek için Microfil kullanmış olduğu bir döküm tekniği açıklar. Iskemi ya da şiddeti seviyesi arteriyel bağlanma / eksizyonu yapılır yere göre değişir. Biz profunda femoris ve epigastrikan arterlerin kökeni proksimal femoral arter bir çift ligasyonu oluşturulan, bu yaklaşımın distal b...

Periferik arter hastalığı (PAH) ve bacaklar 4 yetersiz kan akımı neden olan bir aterosklerotik hastalıktır. 8 ile 12 milyon Amerikalı etkiler ve mevcut tıbbi tedaviler sadece sınırlı rahatlama 5,6 sunuyoruz. Bacaklarda kan dolaşımını artırmak terapötik ajanlar sadece hastalığın ilerlemesini engellemek değil, aynı zamanda yaşam kalitesini artırmak olmaz. Genellikle yaşlı hastalarda görülen düşük böbrek ve karaciğer fonksiyon ilaç metabolizması azaltmak ve sistemik yönetimi ile yan etkileri artırabilir çünkü PAD insidansı 50 yaş üzerindeki kişilerde daha yüksektir, bu nedenle yerel farmakolojik tedavi daha arzu edilen bir tedavi yöntemidir. Böylece, yerel olarak yönetilen ajanlar anjiyogenez ve mikrovasküler yeniden teşvik ederek bacak iskemi hafifletmek olmadığını incelemek için PAD bir fare modeli yarattı. Özellikle de, sürekli olarak terapötik maddeyi sunmak için bir kateter takılmıştır ozmotik pompa kullanıldığındafarelerin iskemik uyluk kas. Bizim dağıtım sistemi kullanarak, yerel ortamda ilacın optimum konsantrasyonu korumak başardık, bu yaklaşım, uygun ilaç biyoaktivitesi sağlar olası sistemik yan etkileri önler ve sistemik yönetimi ile ilgili sınırlı yerel uyuşturucu erişim dezavantajı üstesinden gelir. Ayrıca, yerel olarak yönetilen ajanlar revaskülarizasyon teşvik olmadığını değerlendirmek için, ileri döküm ve mikrovasküler değişiklikler ölçümü sağlayan yüksek çözünürlüklü görüntüleme teknikleri kullanılır. Bununla birlikte, bu video makalede kullanılan metotlar kombinasyonu PAD hastalarda 7-9 farmakolojik revaskülarizasyon anlaşılmasına yardımcı olmak üzere ön-klinik çalışmalarda yararlıdır.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

Periferik arter hastalığı olan hayvan modellerinde ön-klinik araştırma mikro teşvik etmek için tasarlanmış terapötik maddelerin etkinliğinin test edilmesinde bir rol oynar. Uygulama yoluna derinden bu ajanların 1,2 biyo ve etkinliğini etkiler, çünkü bu maddeler için dağıtım yöntemi seçimi önemlidir. Bu makalede, yerel bir kateterize ozmotik pompa kullanarak iskemik arka ayaklarında bir madde yönetmek nasıl gösterilmektedir. Bu pompa uzun bir süre boyunca sürekli olarak ayrılan sulu bir solüsyonunun bir şekilde sabit hacimli bir teslim edebilir. Ayrıca sol bacak içinde profunda femoris ve epigastrikan arterlerin kökeni proksimal femoral arter ligasyonu ile oluşturulan tek taraflı bacak iskemi bizim fare modeli sunuyoruz. Son olarak, Microfil, bir silikon radyoopak döküm maddesi ile infrarenal abdominal aorta ve bacak damar perfüzyon in vivo kanülasyon ve ligasyonu tarif. Microfil serpmek ve dolgu tüm vasküler yatak (arteriyel ve venöz), ve çıkış için büyük damar kablo bağlandı çünkü, ajan küçük numune mikro-CT 3 kullanımı ile gelecek ex vivo görüntüleme için damar muhafaza edilebilir olabilir .

Animasyon protokol ayrıntılı ozmotik pompa ekleme ve bacak iskemi cerrahi göstermektedir. Şekil 1 iskemi teyit hindlimb perfüzyon lazer Doppler görüntüleri gösterir. Microfil vasküler Döküm işleminden sonra, damar ağının bir 3D mikro CT görüntüsünde Microfil (Şekil 2A) etkin gemiler doldurabileceği gösterir, ancak bu süreksizlik (örneğin, hava kabarcıkları, eksikliği çeşitli usule faktörlere bağlı olarak (Şekil 2B) oluşabili...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

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

Biz burada göstermek<em&gt; In vivo</emSürekli yerel ilaç dağıtım ve bir fare modelinde, bacak iskemisi oluşturulması için bir ozmotik pompa&gt; yerleştirilmesi. Ayrıca, bacak damar mikro-bilgisayarlı tomografi (mikro-BT) görüntüleme için hazırlamak için, Microfil, bir silikon radyoopak madde ile perfüze.

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 ...

osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Research

JoVE Journal

Medicine

12.2K Görüntüleme.  The Texas Heart Institute at St. Luke's Episcopal Hospital. Biz burada göstermek In vivo yerleştirilmesi. Ayrıca, bacak damar mikro-bilgisayarlı tomografi (mikro-BT) görüntüleme için hazırlamak için, Microfil, bir silikon radyoopak madde ile perfüze.

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

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

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

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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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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Fare Arteriovenöz Fistül Endotelyumunun için intraluminal Drug Delivery

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Bir Sıçan Karaciğer Hilar Kelepçe Modeli Kullanılarak Doğrudan Segmental İçi hepatik Dağıtım Yöntemi

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor, büyük metabolitleri ve Lumacaftor Kistik fibrozis hastalarının biyolojik sıvı içinde klinik örnekler yüksek üretilen iş analizi için en iyi duruma getirilmiş LC-MS/MS yöntemi

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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.