微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装
Summary
冠状动脉血管的可视化来推进我们的心血管疾病的认识是至关重要的。在这里，我们描述了在微型计算机断层扫描成像（μCT）准备，与不透X线的硅橡胶（Microfil）灌注小鼠冠状动脉血管的方法。
Abstract
了解许多不同的疾病状态的可视化的血管变得越来越重要。虽然一些技术成像血管存在，很少有能够可视化作为一个整体的血管网，同时延伸到了一项决议，包括较小的船只1,2。此外，许多血管铸型技术破坏周围组织，防止进一步分析样品3-5。规避这些问题的方法之一是微型计算机断层扫描（μCT）。 μCT成像扫描分辨率<10微米，是能够产生三维重建血管网，并留下完整的后续分析组织（例如，组织学和形态学）6-11。然而，通过体外 μCT方法成像船只需要，船只可与不透X线的化合物填充。 μCT成像产生血管的准确表示是因为如此，然后船只的可靠和完整的灌装。在这个协议中，我们描述了μCT成像小鼠冠状血管，准备灌装技术。
两个占主导地位的技术填补了冠状动脉血管存在： 在体内通过主动脉插管和逆行灌注（或关闭主动脉弓分支）12-14日 ，或通过Langendorff灌流系统15-17 体外 。在这里，我们描述了在体内主动脉插管的方法已被专门设计，以确保填写所有船只。我们使用低粘度不透X线的化合物称为Microfil填补所有的毛细血管，以及动脉和静脉血管网双方可以通过最小血管灌注。血管灌注使用加压灌注系统的缓冲，然后填写Microfil。为了确保Microfil填补了小的阻力血管，结扎大枝emanating从主动脉，转移到冠状动脉Microfil。一旦填写完成后，挤压Microfil了一些船只，以防止心脏组织的弹性性质，我们结扎灌装后立即访问的主要血管的出口点。因此，我们的技术进行了优化，完整的充填和充填剂的最大保留，使完整的冠状动脉血管网的可视化 – 动脉，毛细血管和静脉的一致好评。
Protocol
1。开始前的准备填写与血管舒张缓冲区（4mg / L的罂粟碱+ 1g / L的腺苷在PBS）或4％多聚甲醛（PFA）在PBS压力灌注装置两侧分别。 准备填补1:100肝素0.1毫升（5000U/ml股票）和弯曲针〜120度角的斜面向上1 1/2cc胰岛素注射器（带一个永久连接的29G半“针）。做用1毫升注射器装满0.3毫升饱和氯化钾溶液（1 26G半“针）相同。 2。暴露心脏插管主动脉使用您选?…
Discussion
心脏组织有一个非常高的代谢需求，因此需要不断交付的冠状动脉血管的血液供应养分和氧气。疾病的冠状血管，降低因血管狭窄和阻塞的冠状动脉功能，可导致组织缺血缺氧，并把受影响的病人，心肌梗死和心脏肌肉造成不可弥补的损害的风险。一个更好地了解这些船只的疾病状态是必要的，关键我们的学习能力冠状动脉血管的可视化。在这里，我们提出了一个准备前体内成像小鼠冠状?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢凯利·史蒂文斯博士的协议，迈克尔·西蒙斯博士，博士硖Hauch，一般性讨论实验室成员的初步试验。
这项工作是由NIH资助HL087513和P01号HL094374支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| 1 ml syringes | Becton Dickinson | BD-309602 | |
| 1/2cc insulin syringes with permanently attached 29G ½’ needles | Becton Dickinson | BD-309306 | |
| 2″ x 2″ Gauze pads | Med101store.com | SKU 2208 | |
| 24G ¾” Angiocath IV catheter | Becton Dickinson | BD-381112 | |
| 26G ½”gauge needles | Becton Dickinson | BD-305111 | |
| Adenosine | Sigma | A9251 | 1g/L in PBS for Vasodilation Buffer (with Papaverine) |
| Angled Graefe Forceps | Fine Science Tools | 11052-10 | |
| Cotton-tipped applicators: 6″ non-sterile | Cardinal Health | C15055-006 | |
| Curved Surgical Scissors | Fine Science Tools | 14085-09 | |
| Dissecting stereoscope and light source | Nikon | NA | NA |
| Dissecting Tray, 11.5 x 7.5 inches | Cole-Parmer | YO-10915-12 | Filled with tar for pinning down the mouse |
| Fine Curved Forceps | Aesculap | FD281R | Need two |
| Heparin, 5000 U/ml stock | APP Pharmaceuticals LLC | NDC 63323-047-10 | 1:100 dilution in water |
| KCl | Fisher | P217 | Saturated solution in H2O |
| Ketamin (Ketaset), 100 mg/ml stock | Fort Dodge, Overland Park, KS, USA | NDC 0856-2013-01 | Mixed as 130 mg/kg body weight, with Xylazine in 0.9% saline |
| Microfil | Flow Tech | MV-122 (yellow). Other color options are also available. | Mix 1:1 by weight, with 10% by volume of curing agent. Prepare just before injection, and vortex to ensure it is well mixed |
| Non-sterile Suture: 6-0, braided silk | Harvard Apparatus | 723287 | |
| Papaverine | American Regent Inc. | NDC 0517-4010-01 | 4mg/L in PBS for Vasodilation Buffer (with Adenosine) |
| Paraformaldehyde | Sigma | P6148 | Prepared as 4% solution |
| Perfusion Apparatus | See figure 2 | ||
| Spring Scissors | Fine Science Tools | 15018-10 | |
| Xylazine (Anased), 20 mg/gl stock | Lloyd Labs | NADA #139-236 | Mixed as 8.8 mg/kg body weight, with Ketamin in 0.9% saline |
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Tags
Retrograde PerfusionFillingMouse Coronary VasculatureMicro Computed Tomography ImagingVisualizationImaging TechniquesVascular NetworkResolutionVascular Casting TechniquesMicro-Computed Tomography (µCT)Radiopaque CompoundAccurate RepresentationFilling TechniqueIn Vivo CannulationRetrograde PerfusionLangendorff Perfusion System
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Weyers, J. J., Carlson, D. D., Murry, C. E., Schwartz, S. M., Mahoney, Jr., W. M. Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging. J. Vis. Exp. (60), e3740, doi:10.3791/3740 (2012).