Ringraziamo il Dott. Kelly Stevens per le prove iniziali del protocollo, il dottor Michael Simons, Hauch Kip Dr., e membri di entrambi i loro laboratori per la discussione generale. Questo lavoro è l&#39;assistenza di sovvenzioni NIH HL087513 e P01 HL094374.

1. Preparativi prima di iniziare <ol><li> Riempire ciascun lato dell&#39;apparecchio perfusione pressione con tampone Vasodilator (4 mg / L papaverina + 1 g / L adenosina in PBS) o 4% paraformaldeide (PFA) in PBS, rispettivamente. </li><li> Preparare una siringa 1/2cc insulina (con una 29G permanentemente attaccato ½ &quot;needle), riempiendolo con 0,1 ml di eparina 1:100 (stock 5000U/ml) e piegando l&#39;ago ~ 120 gradi con il smussata. Do the stesso con una siringa da 1 ml (con 26G ½ &quot;spillo) riempita con 0,3 ml di soluzione satura di KCl. </li></ol> 2. L&#39;esposizione del cuore e dell&#39;aorta cannulating <ol><li> Anestetizzare il mouse utilizzando il anestetico di scelta. (Usiamo una dose eccessiva di una miscela di ketamina / Xylazina:. IP iniezione di 130 mg / kg di ketamina e 8,8 mg / kg Xylazina in soluzione salina) </li><li> Appunta il mouse anestetizzato sul vassoio dissezione, il lato ventrale. Aprire la cavità addominale con un&#39;incisione mediana e ritrarre la pelleper esporre gli organi. Spostare l&#39;intestino da un lato per esporre una regione del posteriore Vena Cava (PVC). </li><li> Iniettare la soluzione di eparina in PVC. Come si estrae l&#39;ago, coprire il foro dell&#39;ago con un cotton-applicatore con la punta per evitare perdite e tenerlo premuto per alcuni secondi fino a quando i coaguli di muro in PVC e guarnizioni. Attendere 2-3 minuti per l&#39;eparina per disperdere tutta la circolazione del mouse. </li><li> Sezionare il diaframma e la gabbia toracica in modo da poter osservare il cuore che batte. Iniettare lentamente soluzione KCl in PVC finché gli arresti cardiaci. </li><li> Rimuovere tutti gli organi sotto il diaframma e accise, la porzione posteriore del mouse, lasciando la regione anteriore del diaframma intatto. Rimuovere il diaframma, avendo cura di tagliare il PVC in prossimità del diaframma porzione prossimale al cuore è facile individuare in fasi successive. </li><li> Individuare l&#39;estremità tagliata dell&#39;aorta. Posizionare una lunghezza lunga di 6-0 sutura di seta intrecciata sotto l&#39;aorta di pochi millimetri anteriori from l&#39;estremità tagliata in modo tale che la sutura è raddoppiata su se stesso. Tagliare questa sutura a metà in modo più ci sono 2 pezzi di sutura sotto l&#39;aorta. Inserire il angiocatheter nell&#39;estremità taglio dell&#39;aorta (Fig. 1A, B) e collegare ogni sutura con un doppio nodo per tenere il angiocatheter in posizione e impedire qualsiasi contropressione all&#39;interno della aorta fuoriuscita. </li></ol> 3. Perfusione e iniezione Microfil <ol><li> Collegare il angiocatheter all&#39;apparecchio perfusione pressione (Fig. 2) e iniziare la perfusione dei vasi con tampone vasodilatatore (Fig. 1C) pompando l&#39;apparecchio perfusione ad una pressione di guida 100-110 mmHg. Doppio controllare che buffer viene perfusione attraverso le coronarie, garantendo liquido è uscita dal PVC. Continuare a perfusione per almeno 3 minuti o fino a quando il fluido che esce il PVC è chiaro. (Continua con le fasi successive, mentre perfusione.) </li><li> Staccare le costole e schiena pin (o rimuovere) la gabbia toracica per esporre il cuore. Dopo l&#39;esposizione, essere cacon cautela e precisione per non lasciare che il cuore asciugare dalla spremitura gocce di tampone sul cuore da un buffer imbevuta di pezzo di garza. Sgombrare il timo per esporre l&#39;arco aortico. Legare i tre rami principali dell&#39;aorta 6-0 con sutura intrecciata in seta per garantire il fluido viene deviata attraverso le coronarie piuttosto che attraverso questi, i più grandi vasi a bassa resistenza (Fig. 1D). </li><li> Perfusione del cuore con fissativo per 15 minuti, quindi risciacquare con tampone vasodilatazione per almeno 2 minuti. Nel frattempo, legare sia anteriore vena cava per impedire Microfil di fuoriuscire dal cuore dopo l&#39;iniezione (Figura 1E). Suture Luogo in tutto il PVC e l&#39;aorta, ma non li stringono fino a dopo il riempimento. </li><li> Preparare la Microfil (come specificato nella tabella dei reagenti) e caricarlo in una siringa da 1 ml. Riempire la vaschetta dissezione con acqua sufficiente a coprire il catetere (in modo da impedire l&#39;introduzione di bolle d&#39;aria quando si passa dalla tubazione perfusione alla siringa Microfil). Scollegare la perfusione apparatus dal catetere e collegare la siringa Microfil. </li><li> Iniettare il Microfil nell&#39;aorta fino buon riempimento delle arterie coronarie è evidente (figure 1F, 3A): le arterie si riempirà prima, e poi la si Microfil &quot;cadere&quot; nei capillari come le vampate di tessuto con il colore della Microfil. Una volta sul lato venoso, la natura idrofoba del Microfil causa di apparire inizialmente come sfere indipendenti come emerge dai vasi più piccoli. Continuare ad iniettare il Microfil finché una colonna continua riempie le vene. Riempimento completo sarà evidente quando il Microfil è continua all&#39;interno dei vasi, e si esce attraverso il PVC. </li><li> Dopo il riempimento è completata, rapidamente serrare le suture precedentemente disposte intorno al PVC e aorta per impedire la natura elastica del tessuto cardiaco dalla spremitura del Microfil fuori dei vasi. </li><li> Coprire il cuore con una garza umida (inzuppato d&#39;acqua dal vassoio di dissezione) Per evitare che si secchi, e lasciate riposare per circa 1 ora a temperatura ambiente fino a quando il Microfil è polimerizzato. Evitare pressioni esterne sul cuore durante il processo di polimerizzazione, come sollevamento o ruotando il cuore nel tentativo di ottenere una vista precoce dei vasi pieni nella parte posteriore del cuore. Questo può spremere Microfil da alcuni vasi in una zona più elastica del cuore, causando rotture nel Microfil. </li><li> Rimuovere il cuore e post-fix in 4% PFA notte a 4 ° C. Poi memorizzare in etanolo 70% a 4 ° C. La vascolarizzazione cardiaca è ora pronto per l&#39;imaging μCT. </li></ol> 4. Risultati rappresentativi Le navi che vengono effettivamente irrorate da Microfil avrà continua, ininterrotta per tutta Microfil i vasi (Fig. 3A). Il grado di riempimento dei vasi coronarici può essere giudicato da occhio; vene si trovano epicardially 18, e può essere facilmente osservato (Fig. 3A, freccia); arterie,che sono più intramiocardico 18, sono anche visibili attraverso la superficie del cuore (Fig. 3A, freccia). Riempimento capillare è anche evidente, come tessuto cardiaco ha una elevata densità di capillari, e pertanto, quando il riempimento capillari, il tessuto cardiaco si filo con il colore del Microfil (Fig. 3A, stella). Così, le reti vascolari che non hanno riempire sarà evidente a causa della mancanza di Microfil (Fig. 3B, C). Discontinuità nella Microfil (asterischi in Fig. 3B) appaiono spesso perché la natura idrofoba del Microfil lo farà a contrarsi in sé e provocare &quot;infrange&quot; all&#39;interno dei vasi riempiti. Questi &quot;pause&quot; può essere ridotta se la pressione all&#39;interno dei vasi è mantenuta attraverso adeguata tie-off dei punti di uscita vascolari dal cuore. Altre discontinuità può essere causato da bolle d&#39;aria all&#39;interno del Microfil. Per evitare l&#39;introduzione di aria, assicurarsi che il angiocatheter è completamente immerso in acqua quando si passa dalla perfusione ap<html> Paratus alla siringa Microfil. Se una bolla d&#39;aria viene introdotta, spesso può essere rimosso semplicemente continuando la perfusione Microfil finché la bolla è stato spinto attraverso e fuori dei vasi coronarici. Reti vascolari non può riempire completamente se una porzione del letto vascolare è bloccato (Fig. 3B, freccia). Mentre eparina inibisce la formazione di coaguli di sangue, blocchi occasionali possono ancora verificarsi a causa di perfusione eparina incomplete prima di iniziare la procedura, oa causa di altri fattori sconosciuti. Se si verifica un blocco, non vi è, a nostra conoscenza, nessun metodo per staccare il blocco per completare il riempimento vascolare. Riempimento incompleto può anche causare una pressione troppo poco viene utilizzato durante il riempimento, la Microfil non sarà costretto a tutti i letti vascolari e le reti capillari (Fig. 3C). Al contrario, una pressione eccessiva può causare i capillari a scoppiare e estravasare Microfil nel tessuto circostante (Fig. 3D). files/ftp_upload/3740/3740fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Panoramica del sistema di perfusione Microfil. (A) L&#39;aorta e il PVC vengono tagliati approssimativamente a livello del diaframma. (B) L&#39;aorta ascendente è cannulata con uno angiocatheter. (C) buffer vasodilatazione è perfuso attraverso i vasi, spinti dalla pressione di perfusione l&#39;apparato (non raffigurato), mentre (D) i tre rami principali fuori l&#39;arco aortico vengono legati. (E) 4% PFA è perfuso attraverso le coronarie, mentre entrambe le anteriori Vena Cavas sono legato. (F) usando una siringa, Microfil viene perfuso attraverso le coronarie finché si osserva in uscita dal PVC. <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="Figura 2"/> Figura 2. Apparecchiatura di perfusione. Due beute, ciascuna riempita di tampone vasodilatazione o 4% PFA, sono uniti e pressurizzato attraverso tubi collegati alle loro armi bianche. Il sistema viene pressurizzato mediante pompaggio manuale deibulbo, e un manometro è collegato ad uno dei flaconi per consentire il monitoraggio e il mantenimento della pressione. Piccoli tubi si estendono attraverso tappi di gomma e giù nel fluido in ogni contenitore. Pressione in entrata dalla pompe armi bianche del fluido da ciascuna beuta questi tubi più piccoli. I tubi quindi unire ad un rubinetto che consente solo passaggio del fluido da un pallone alla volta. <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="Figura 3"/> Figura 3. Assaggiate cuori microriempiti. (A) Le navi che vengono riempite ben avranno pochi (se del caso) Soggiorni in Microfil, e il tessuto cardiaco sarà tinge il colore del Microfil a causa della piena capillari (a stella, e si confrontano con C). Entrambe le arterie (freccia - dell&#39;arteria discendente anteriore sinistra) e le vene (punta di freccia - Sinistra Vein coronarica) sono visibili attraverso la superficie del cuore. (B) Un cuore con interruzioni nel Microfil (asterischi) e blocchi in alcuni vasi che impedito Mic completorofil penetrazione. I vasi rimangono bloccati rosso (freccia), come il sangue non è stata lavata durante il processo di perfusione. (C) Un cuore con le navi che sono stati completamente riempiti. Si noti il ​​tessuto non ha preso il colore giallo del Microfil, indicando il Microfil non penetrano nei capillari. (D) Un cuore in cui il burst capillari durante il riempimento, causando la Microfil di perdite nel tessuto circostante (freccia).

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	<li>Couffinhal, T., Dufourcq, P., Barandon, L., Leroux, L., Duplaa, C. <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+to+study+angiogenesis+in+the+context+of+cardiovascular+diseases.">Mouse models to study angiogenesis in the context of cardiovascular diseases.</a> Front. Biosci. 14, 3310-3325 (2009).</li><li>Zagorchev, L., Mulligan-Kehoe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+vessels+in+mouse+models+of+disease.">Molecular imaging of vessels in mouse models of disease.</a> Eur. J. Radiol. 70, 305-311 (2009).</li><li>Krucker, T., Lang, A., Meyer, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+polyurethane-based+material+for+vascular+corrosion+casting+with+improved+physical+and+imaging+characteristics.">New polyurethane-based material for vascular corrosion casting with improved physical and imaging characteristics.</a> Microsc. Res. Tech. 69, 138-147 (2006).</li><li>Murakami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+patterns+in+the+rat+pancreas:+a+simulative+demonstration+by+injection+replication+and+scanning+electron+microscopy.">Blood flow patterns in the rat pancreas: a simulative demonstration by injection replication and scanning electron microscopy.</a> Microsc. Res. Tech. 37, 497-508 (1997).</li><li>Icardo, J. M., Colvee, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+course+of+the+coronary+arteries+in+normal+mice+and+in+iv/iv+mice.">Origin and course of the coronary arteries in normal mice and in iv/iv mice.</a> J. Anat. 199, 473-482 (2001).</li><li>Beighley, P. E., Thomas, P. J., Jorgensen, S. M., Ritman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+architecture+of+myocardial+microcirculation+in+intact+rat+heart:+a+study+with+micro-CT.">3D architecture of myocardial microcirculation in intact rat heart: a study with micro-CT.</a> Adv. Exp. Med. Biol. 430, 165-175 (1997).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R1267-R1279 (2002).</li><li>Jorgensen, S. M., Demirkaya, O., Ritman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+vasculature+and+parenchyma+in+intact+rodent+organs+with+X-ray+micro-CT.">Three-dimensional imaging of vasculature and parenchyma in intact rodent organs with X-ray micro-CT.</a> Am. J. Physiol. 275, H1103-H1114 (1998).</li><li>Marxen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Med. Phys. 31, 305-313 (2004).</li><li>Zagorchev, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro+computed+tomography+for+vascular+exploration.">Micro computed tomography for vascular exploration.</a> J. Angiogenes. Res. 2, 7-7 (2010).</li><li>Heinzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+microimaging+for+multiscale+analysis+of+large+vascular+networks.">Hierarchical microimaging for multiscale analysis of large vascular networks.</a> Neuroimage. 32, 626-636 (2006).</li><li>Dedkov, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synectin/syndecan-4+regulate+coronary+arteriolar+growth+during+development.">Synectin/syndecan-4 regulate coronary arteriolar growth during development.</a> Dev. Dyn. 236, 2004-2010 (2007).</li><li>Gossl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+and+hemodynamic+characteristics+of+vasa+vasorum+in+the+walls+of+porcine+coronary+arteries.">Functional anatomy and hemodynamic characteristics of vasa vasorum in the walls of porcine coronary arteries.</a> Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 272, 526-537 (2003).</li><li>Rodriguez-Porcel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+myocardial+microvascular+3D+architecture+in+experimental+hypercholesterolemia.">Altered myocardial microvascular 3D architecture in experimental hypercholesterolemia.</a> Circulation. 102, 2028-2030 (2000).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+The+Langendorff+technique+of+isolated+heart+perfusion.">Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion.</a> J. Mol. Cell. Cardiol. 50, 940-950 (2011).</li><li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J. Pharmacol. Toxicol. Methods. 55, 113-126 (2007).</li><li>Toyota, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+is+required+for+coronary+collateral+growth+in+the+rat.">Vascular endothelial growth factor is required for coronary collateral growth in the rat.</a> Circulation. 112, 2108-2113 (2005).</li><li>Lavine, K. J., Long, F., Choi, K., Smith, C., Ornitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+to+distinct+cell+types+differentially+regulates+coronary+artery+and+vein+development.">Hedgehog signaling to distinct cell types differentially regulates coronary artery and vein development.</a> Development. 135, 3161-3171 (2008).</li><li>Cheema, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adventitial+microvessel+formation+after+coronary+stenting+and+the+effects+of+SU11218,+a+tyrosine+kinase+inhibitor.">Adventitial microvessel formation after coronary stenting and the effects of SU11218, a tyrosine kinase inhibitor.</a> J. Am. Coll. Cardiol. 47, 1067-1075 (2006).</li><li>Lametschwandtner, A., Lametschwandtner, U., Weiger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+vascular+corrosion+casts--technique+and+applications:+updated+review.">Scanning electron microscopy of vascular corrosion casts--technique and applications: updated review.</a> Scanning Microsc. 4, 889-941 (1990).</li><li>Schneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+3D+visualization+and+quantification+of+murine+bone+and+bone+vasculature+using+micro-computed+tomography+and+vascular+replica.">Simultaneous 3D visualization and quantification of murine bone and bone vasculature using micro-computed tomography and vascular replica.</a> Microsc. Res. Tech. 72, 690-701 (2009).</li><li>Manelli, A., Sangiorgi, S., Binaghi, E., Raspanti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+analysis+of+SEM+images+of+corrosion+casting+using+adaptive+stereo+matching.">3D analysis of SEM images of corrosion casting using adaptive stereo matching.</a> Microscopy Research and Technique. 70, 350-354 (2007).</li><li>Alanentalo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat. Meth. 4, 31-33 (2007).</li><li>Quintana, L., Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical projection tomography of vertebrate embryo development. , 586-594 (2011).</li><li>Walls, J. R., Coultas, L., Rossant, J., Henkelman, 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=Three-Dimensional+Analysis+of+Vascular+Development+in+the+Mouse+Embryo.">Three-Dimensional Analysis of Vascular Development in the Mouse Embryo.</a> PLoS ONE. 3, e2853-e2853 (2008).</li><li>Chalothorn, D., Clayton, J. A., Zhang, H., Pomp, D., Faber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+density,+remodeling,+and+VEGF-A+expression+differ+widely+between+mouse+strains.">Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.</a> Physiol. Genomics. 30, 179-191 (2007).</li><li>Behm, C. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Endothelial+Vascular+Cell+Adhesion+Molecule-1+Expression+and+Inflammatory+Cell+Recruitment+During+Vasculogenesis+and+Ischemia-Mediated+Arteriogenesis.">Molecular Imaging of Endothelial Vascular Cell Adhesion Molecule-1 Expression and Inflammatory Cell Recruitment During Vasculogenesis and Ischemia-Mediated Arteriogenesis.</a> Circulation. 117, 2902-2911 (2008).</li><li>Carr, C. L., Lindner, J. 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>

I topi sono stati trattati con metodi approvati dalla Institutional Animal Care e del Comitato uso della University of Washington e in conformità con la Guida per la cura e l&#39;uso di animali da laboratorio pubblicato dal US National Institutes of Health (NIH Publication No. 85-23, riveduta nel 1996).

Tessuto cardiaco ha una forte domanda metabolica, e richiede pertanto un costante rifornimento di sostanze nutritive e ossigeno dal sangue erogata dal sistema vascolare coronarico. Malattie dei vasi coronarici, che riducono la funzione a causa di stenosi coronarica nave e blocco, possono portare a ipossia tissutale e di ischemia, e mettere i pazienti affetti a rischio di infarto miocardico e danni irreparabili al muscolo cardiaco. Una migliore comprensione dello stato di malattia di questi vasi è necessario, e critico ...

<html><table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 1 ml siringhe </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309602 </td><td></td></tr><tr><td> Siringhe da insulina 1/2cc con aghi fissi ½ 29G &#39; </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309306 </td><td></td></tr><tr><td> Garza 2 &quot;x 2&quot; pastiglie </td><td> Med101store.com </td><td> SKU 2208 </td><td></td></tr><tr><td> 24G ¾ &quot;Angiocath IV catetere </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-381112 </td><td></td></tr><tr><td> 26G ½ &quot;aghi calibro </td><td>lank &quot;&gt; Becton Dickinson </td><td> BD-305111 </td><td></td></tr><tr><td> L&#39;adenosina </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> A9251 </td><td> 1 g / L in PBS per Buffer Vasodilatazione (con papaverina) </td></tr><tr><td> Pinze angolari Graefe </td><td> <a href="http://www.finescience.com/" target="_blank">Strumenti di Scienza Fine</a> </td><td> 11052-10 </td><td></td></tr><tr><td> Cotton-punte: 6 applicatori &quot;non sterile </td><td> <a href="http://www.cardinal.com/" target="_blank">Cardinal Health</a> </td><td> C15055-006 </td><td></td></tr><tr><td> Forbici chirurgiche curve </td><td> <a href="http://www.finescience.com/" target="_blank">Strumenti di Scienza Fine</a> </td><td> 14085-09 </td><td></td></tr><tr><td> Stereoscopio dissezione e la sorgente di luce </td><td> <a href="http://www.nikon.com/" target="_blank">Nikon</a> </td><td> NA </td><td> NA </td></tr><tr><td> Vassoio dissezione, 11,5 x 7,5 pollici </td><td> <a href="http://www.coleparmer.com/" target="_blank">Cole-Parmer</a> </td><td> YO-10.915-12 </td><td> Riempito con tar per appuntare premuto il mouse </td></tr><tr><td> Fine pinze curve </td><td> <a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a> </td><td> FD281R </td><td> Hai bisogno di due </td></tr><tr><td> L&#39;eparina, 5000 U / ml magazzino </td><td> <a href="http://www.apppharma.com/" target="_blank">APP Pharmaceuticals LLC</a> </td><td> NDC 63323-047-10 </td><td> Diluizione 1:100 in acqua </td></tr><tr><td> KCl </td><td> <a href="http://www.fishersci.com/" target="_blank">Pescatore</a> </td><td> P217 </td><td> Soluzione satura di H2O </td></tr><tr><td> Ketamina (Ketaset), 100 mg / ml magazzino </td><td> Fort Dodge, Overland Park, KS, USA </td><td> NDC 0856-2013-01 </td><td> Misto di 130 mg / kg di peso corporeo, con Xylazina allo 0,9% sAline </td></tr><tr><td> Microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">Flusso Tech</a> </td><td> MV-122 (giallo). Altre opzioni di colore sono inoltre disponibili. </td><td> Mescolare 01:01 in peso, con il 10% in volume di catalizzatore. Preparare immediatamente prima dell&#39;iniezione, e vortex per assicurarsi che sia ben amalgamato </td></tr><tr><td> Non sterile di sutura: 6-0, seta intrecciata </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">Harvard Apparatus</a> </td><td> 723287 </td><td></td></tr><tr><td> Papaverina </td><td> <a href="http://www.americanregent.com/" target="_blank">Americano Regent Inc.</a> </td><td> NDC 0517-4010-01 </td><td> 4 mg / L in PBS per Buffer Vasodilatazione (con adenosina) </td></tr><tr><td> Paraformaldeide </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> P6148 </td><td> Preparato come soluzione al 4% </td></tr><tr><td> Perfusione Apparatus </td><td></td><td></td><td> Vedere la figura 2 </td></tr><tr><td> Primavera Forbici </td><td> <a href="http://www.finescience.com/" target="_blank">Strumenti di Scienza Fine</a> </td><td> 15018-10 </td><td></td></tr><tr><td> Xilazina (Anased), 20 mg / gl magazzino </td><td> <a href="http://www.lloydlab.com/" target="_blank">Lloyd Labs</a> </td><td> NADA # 139-236 </td><td> Misto di 8,8 mg / kg di peso corporeo, con ketamina in soluzione salina 0,9% </td></tr></tbody></table>

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

Visualizzazione della vascolarizzazione sta diventando sempre più importante per capire molti stati patologici diversi. Mentre esistono diverse tecniche per l&#39;imaging vascolare, pochi sono in grado di visualizzare la rete vascolare nel suo insieme, mentre si estende a una risoluzione che include il 1,2 imbarcazioni più piccole. Inoltre, molte tecniche di fusione vascolari distruggere il tessuto circostante, impedendo ulteriori analisi del campione 3-5. Un metodo che elude questi temi è micro-Tomografia Computerizzata (μCT). μCT possibile eseguire la scansione di immagini con risoluzione inferiore a 10 micron, è capace di produrre ricostruzioni 3D della rete vascolare, e lascia intatto il tessuto per l&#39;analisi successiva (ad esempio, l&#39;istologia e la morfometria) 6-11. Tuttavia, navi imaging di ex vivo metodi μCT richiede che i vasi da riempire con un composto radiopaco. Come tale, la rappresentazione accurata di vasi prodotta da μCT immagini è subordinatoriempimento affidabile e completa dei vasi. In questo protocollo, si descrive una tecnica per il riempimento di vasi coronarici del mouse in preparazione per l&#39;imaging μCT. Due tecniche predominano esistono per il riempimento del sistema vascolare coronarico: in vivo via cannulazione e perfusione retrograda dell&#39;aorta (o di un ramo dell&#39;arco aortico) 12-14, o ex vivo mediante un sistema di perfusione Langendorff 15-17. Qui si descrive un metodo in cannulazione aortica vivo che è stato specificamente progettato per garantire il riempimento di tutte le navi. Usiamo un composto a bassa viscosità radiopaco chiamato Microfil che può perfondere attraverso i piccoli vasi per riempire tutti i capillari, nonché entrambi i lati arterioso e venoso della rete vascolare. Le navi sono perfusi con tampone utilizzando un sistema di pressione di perfusione, e poi riempito con Microfil. Per garantire che Microfil riempie i piccoli vasi di resistenza superiori, abbiamo legare i rami di grandi dimensioni emanating dall&#39;aorta, che devia il Microfil nelle coronarie. Una volta che il riempimento è completata, per impedire la natura elastica del tessuto cardiaco da spremere fuori Microfil di alcuni vasi, si legare accessibili i punti importanti uscita vascolari immediatamente dopo il riempimento. Pertanto, la nostra tecnica è ottimizzato per il riempimento completo e la ritenzione massimo dell&#39;agente di riempimento, permettendo la visualizzazione della completa rete vascolare coronarico -, capillari, arterie e vene simili.

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

Visualizzazione dei vasi coronarici è fondamentale per avanzare la nostra comprensione delle malattie cardiovascolari. Qui si descrive un metodo per la perfusione vascolare coronarica murino con una gomma di silicone radiopaco (Microfil), in preparazione per le micro-Tomografia Computerizzata (μCT) imaging.

Perfusione retrograda e riempimento del sistema vascolare coronarico mouse come preparazione per la Micro Computed Tomography Imaging

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

retrograde-perfusion-filling-mouse-coronary-vasculature-as

Research

JoVE Journal

Medicine

32.3K Views.  University of Washington. Visualizzazione dei vasi coronarici è fondamentale per avanzare la nostra comprensione delle malattie cardiovascolari. Qui si descrive un metodo per la perfusione vascolare coronarica murino con una gomma di silicone radiopaco (Microfil), in preparazione per le micro-Tomografia Computerizzata (μCT) imaging.

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.

Segmentazione e misurazione dei volumi FAT nei modelli murini di obesità Utilizzo di X-ray Tomografia Computerizzata

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

Ricerca

Medicina

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.

Osmotica Drug Delivery per arti posteriori ischemico e la perfusione dei vasi con Microfil per Micro-Tomografia Computerizzata Imaging

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.

Valutazione della solidità del grasso umano iniezione in topi nudi con Micro-Tomografia Computerizzata

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.

Utilizzando la tomografia micro-computerizzata per la valutazione di sviluppo del tumore e follow-up di risposta al trattamento in un modello murino di cancro ai polmoni

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.

Imaging fluorescente combinata vicino infrarosso e la tomografia micro-computerizzata per Direttamente Visualizzazione cerebrale tromboembolie

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.

Identificazione di calcificazione dell'arteria coronaria su Non-gated scansioni con tomografia computerizzata

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.

Il campionamento continuo del sangue nella tomografia a emissione di positroni di piccole dimensioni/tomografia computerizzata consente la misurazione della funzione di input arteriosa

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.

Tecniche di preparazione dei tessuti per l'imaging micromografico computerizzato con contrasto di modelli cardiaci di mammiferi di grandi dimensioni con malattia cronica

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: Double Resin Casting seguito da Tomografia Micro-Computerizzata per Analisi 3D del Fegato

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.

Preparazione del campione per la visualizzazione tridimensionale basata sulla tomografia computerizzata dei vasi dell'arto posteriore murino

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