We thank Dr. Kelly Stevens for initial trials of the protocol, Dr. Michael Simons, Dr. Kip Hauch, and members of both of their labs for general discussion.
This work is support by NIH grants HL087513 and P01 HL094374.

1. Preparations before starting
<ol> 
 <li>Fill each side of the pressure perfusion apparatus with Vasodilator buffer (4mg/L Papaverine + 1g/L Adenosine in PBS) or 4% Paraformaldehyde (PFA) in PBS, respectively.</li>
 <li>Prepare a 1/2cc Insulin Syringe (with a permanently attached 29G &frac12;" needle) by filling it with 0.1 ml of 1:100 Heparin (5000U/ml stock) and bending the needle to ~120 degree angle with the bevel up. Do the same with a 1 ml syringe (with a 26G &frac12;" needle) filled with 0.3 ml satura...

<ol>
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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. 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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. 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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. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+perfusion+imaging+with+contrast+echocardiography.">Myocardial perfusion imaging with contrast echocardiography.</a> Curr. Cardiol. Rep. 10, 233-239 (2008).</li><li>Leong-Poi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Endogenous+and+Therapeutic+Arteriogenesis+by+Contrast+Ultrasound+Molecular+Imaging+of+Integrin+Expression.">Assessment of Endogenous and Therapeutic Arteriogenesis by Contrast Ultrasound Molecular Imaging of Integrin Expression.</a> Circulation. 111, 3248-3254 (2005).</li><li>Villanueva, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+Targeted+to+Intercellular+Adhesion+Molecule-1+Bind+to+Activated+Coronary+Artery+Endothelial+Cells.">Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells.</a> Circulation. 98, 1-5 (1998).</li><li>Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Myocardial+Blood+Flow+With+Ultrasound-Induced+Destruction+of+Microbubbles+Administered+as+a+Constant+Venous+Infusion.">Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion.</a> Circulation. 97, 473-483 (1998).</li><li>Beckmann, N., Stirnimann, R., Bochelen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Magnetic+Resonance+Angiography+of+the+Mouse+Brain:+Application+to+Murine+Focal+Cerebral+Ischemia+Models.">High-Resolution Magnetic Resonance Angiography of the Mouse Brain: Application to Murine Focal Cerebral Ischemia Models.</a> Journal of Magnetic Resonance. 140, 442-450 (1999).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+MR+angiography+of+intratumoral+vasculature+using+a+novel+macromolecular+MR+contrast+agent.">3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent.</a> Magnetic Resonance in Medicine. 46, 579-585 (2001).</li><li>Nezafat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1-insensitive+T2+preparation+for+improved+coronary+magnetic+resonance+angiography+at+3+T.">B1-insensitive T2 preparation for improved coronary magnetic resonance angiography at 3 T.</a> Magn. 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Cardiac tissue has a very high metabolic demand, and therefore requires a constant supply of nutrients and oxygen from the blood delivered by the coronary vasculature. Diseases of the coronary vessels, which decrease coronary function due to vessel stenosis and blockage, can lead to tissue hypoxia and ischemia, and put affected patients at risk for myocardial infarction and irreparable damage to the heart muscle. A better understanding of the diseased state of these vessels is necessary, and critical to our ability to st...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>1 ml syringes</td>
 <td><a href="http://www.bd.com/" target="_blank">Becton Dickinson</a></td>
 <td>BD-309602</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1/2cc insulin syringes with permanently attached 29G &frac12;' needles</td>
 <td><a href="http://www.bd.com/" target="_blank">Becton Dickinson</a></td>
 <td>BD-309306</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2" x 2" Gauze pads</td>
 <td>Med101store.com</td>
 <td>SKU 2208</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>24G &frac34;" Angiocath IV catheter</td>
 <td><a href="http://www.bd.com/" target="_blank">Becton Dickinson</a></td>
 <td>BD-381112</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>26G &frac12;"gauge needles</td>
 <td><a href="http://www.bd.com/" target="_blank">Becton Dickinson</a></td>
 <td>BD-305111</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Adenosine</td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a></td>
 <td>A9251</td>
 <td>1g/L in PBS for Vasodilation Buffer (with Papaverine)</td>
 </tr>
 <tr>
 <td>Angled Graefe Forceps</td>
 <td><a href="http://www.finescience.com/" target="_blank">Fine Science Tools</a></td>
 <td>11052-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cotton-tipped applicators: 6" non-sterile</td>
 <td><a href="http://www.cardinal.com/" target="_blank">Cardinal Health</a></td>
 <td>C15055-006</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Curved Surgical Scissors</td>
 <td><a href="http://www.finescience.com/" target="_blank">Fine Science Tools</a></td>
 <td>14085-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting stereoscope and light source</td>
 <td><a href="http://www.nikon.com/" target="_blank">Nikon</a></td>
 <td>NA</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>Dissecting Tray, 11.5 x 7.5 inches</td>
 <td><a href="http://www.coleparmer.com/" target="_blank">Cole-Parmer</a></td>
 <td>YO-10915-12</td>
 <td>Filled with tar for pinning down the mouse</td>
 </tr>
 <tr>
 <td>Fine Curved Forceps</td>
 <td><a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a></td>
 <td>FD281R</td>
 <td>Need two</td>
 </tr>
 <tr>
 <td>Heparin, 5000 U/ml stock</td>
 <td><a href="http://www.apppharma.com/" target="_blank">APP Pharmaceuticals LLC</a></td>
 <td>NDC 63323-047-10</td>
 <td>1:100 dilution in water</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td><a href="http://www.fishersci.com/" target="_blank">Fisher</a></td>
 <td>P217</td>
 <td>Saturated solution in H2O</td>
 </tr>
 <tr>
 <td>Ketamin&nbsp; (Ketaset), 100 mg/ml stock</td>
 <td>Fort Dodge, Overland Park, KS, USA</td>
 <td>NDC 0856-2013-01</td>
 <td>Mixed as 130 mg/kg body weight, with Xylazine in 0.9% saline</td>
 </tr>
 <tr>
 <td>Microfil</td>
 <td><a href="http://www.flowtech-inc.com/" target="_blank">Flow Tech</a></td>
 <td>MV-122 (yellow). Other color options are also available.</td>
 <td>Mix 1:1 by weight, with 10% by volume of curing agent. Prepare just before injection, and vortex to ensure it is well mixed</td>
 </tr>
 <tr>
 <td>Non-sterile Suture: 6-0, braided silk</td>
 <td><a href="http://www.harvardapparatus.com/" target="_blank">Harvard Apparatus</a></td>
 <td>723287</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Papaverine</td>
 <td><a href="http://www.americanregent.com/" target="_blank">American Regent Inc.</a></td>
 <td>NDC 0517-4010-01</td>
 <td>4mg/L in PBS for Vasodilation Buffer (with Adenosine)</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td><a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a></td>
 <td>P6148</td>
 <td>Prepared as 4% solution</td>
 </tr>
 <tr>
 <td>Perfusion Apparatus</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>See figure 2</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td><a href="http://www.finescience.com/" target="_blank">Fine Science Tools</a></td>
 <td>15018-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Xylazine (Anased), 20 mg/gl stock</td>
 <td><a href="http://www.lloydlab.com/" target="_blank">Lloyd Labs</a></td>
 <td>NADA #139-236</td>
 <td>Mixed as 8.8 mg/kg body weight, with Ketamin in 0.9% saline</td>
 </tr>
 </tbody>
</table>

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.

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

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.

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

32.2K visualizações.  University of Washington. Visualização dos vasos coronários é fundamental para avançar na compreensão das doenças cardiovasculares. Descrevemos aqui um método para a perfusão vasculatura coronária murino com uma borracha de silicone radiopaco (Microfil), em preparação para imagiologia Tomografia micro-computadorizada (μCT).