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。

1。开始前的准备<ol><li>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。 </li><li>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。 </li></ol> 2。暴露心脏插管主动脉<ol><li>使用您选择的麻醉麻醉鼠标。 （我们使用过量氯胺酮/二甲苯胺噻嗪的混合物:腹腔注射130毫克/千克氯胺酮和8.8毫克/千克盐水甲苯噻嗪） </li><li>引脚上解剖托盘，腹面朝上麻醉鼠标。与正中切口打开腹腔，并收回皮肤暴露的器官。向一侧移动肠子暴露后腔静脉（PVC）的地区。 </li><li>肝素溶液注入的PVC。当你提取针，包括针孔，用棉签涂药，以防止渗漏，保持几秒钟，直到PVC壁血栓和密封。等待2-3分钟肝素驱散整个鼠标流通的。 </li><li>解剖的隔膜和肋骨，所以你可以观察心脏的跳动。慢慢注入KCl溶液在PVC，直到心脏逮捕。 </li><li>删除下面的隔膜和消费的鼠标后部的所有机关，保持膜片地区前不变。取出膜片，小心削减附近的PVC膜片近端心的部分，所以很容易找到，在随后的步骤。 </li><li>找到主动脉截止年底。 1前几毫米来回主动脉下方放置一个长6-0编织丝线米的切端缝合一倍自身。削减在一半不再缝合，所以有2件缝合主动脉。插入主动脉（图1A，B）的截止年底的angiocatheter，并配合双结每个缝合举行到位的angiocatheter和防止主动脉内泄漏出来的任何背压。 </li></ol> 3。灌注Microfil注射的<ol><li>的angiocatheter连接压力灌注装置（图2），并开始灌注到100-110毫米汞柱的驱动压力泵灌注设备，与扩血管缓冲（图1C）的船只。仔细检查缓冲区确保液通过冠状动脉灌注退出从PVC。继续灌注至少3分钟，或直到退出的PVC流体明确。 （继续下一个步骤，而灌流。） </li><li>解剖肋骨和脚背面（或删除）胸腔暴露心脏。一旦暴露，是CAreful不要让心脏干到心脏挤压滴缓冲区，从缓冲区纱布浸湿一块。扫清的胸腺暴露主动脉弓。改通过，而不是通过这些较大，低阻力血管（图1D）的冠状动脉结扎三大主动脉分支，使用6-0编织丝线，以确保流体。 </li><li>灌注固定液15分钟的心脏，然后用清水冲洗至少2分钟舒张缓冲区。同时，结扎两个前腔Cavae Microfil防止泄漏注射后的心脏（图1E）。 PVC和主动脉周围的地方缝合，但不灌装后拧紧，直到他们。 </li><li>准备Microfil（试剂表指定），并装入1 ml注射器。剥离托盘填充足够的水，以弥补导管（以免引入气泡从灌注管时，切换到Microfil注射器）。断开灌注APparatus从导管和连接准备Microfil注射器。 </li><li> Microfil直到注入主动脉冠状动脉良好的填充是显而易见的动脉（图1F，3A）:首先将填补，Microfil"溢出"到毛细血管Microfil的颜色与组织刷新。一旦静脉端，Microfil的疏水性，导致它最初作为独立的领域出现，因为它从较小的船只出现。继续，直到连续列填充静脉注入Microfil。 Microfil是连续的血管内，完整的填充将是显而易见的，它是通过PVC退出。 </li><li>填写完成后，迅速拧紧先前PVC和主动脉周围放置以防止从挤掉船只Microfil心脏组织的弹性性质的缝合线。 </li><li>用湿纱布覆盖的心（从解剖托盘用清水浸泡），以防止其干燥，并让坐在室温约1小时，直到Microfil已聚合。避免对心脏的聚合过程中的任何外部的压力，如解除或转折点的心，试图得到一个早在心脏背面填写船只。这可能从一些船只挤Microfil成的心更弹性的面积，造成的Microfil休息。 </li><li>取出心脏并固定在4％的煤灰发表于4一夜之间°C。然后将其存储在70％乙醇在4°C现在准备μCT成像心脏血管。 </li></ol> 4。代表结果 Microfil有效灌注的容器，将有连续的，整个船只不间断Microfil（图3A）。可以用肉眼来判断冠状动脉血管的充盈程度;静脉epicardially位于18，可以很容易地观察（图3A，箭头）;动脉，这是更心肌内18，也可以通过心脏的表面（图3A，箭头）可见。毛细血管充盈也是显而易见的，如心脏组织的毛细血管的密度非常高，因此，当的毛细血管填充，心肌组织将刷新Microfil的颜色（图3A，明星）。因此，任何未能填补的血管网络将成为Microfil缺乏（图3B，C），由于明显。 Microfil（图3B中的星号）的不连续性，经常出现因为Microfil的疏水性，会导致它收缩到自身造成充满血管内的"休息"。这些"休息"，可减少血管内的压力，如果维持从心脏血管的出口点，通过适当的领带权衡。其他的不连续性，可造成内microfil的气泡。为了防止空气引入，从灌注AP切换时，确保已完全淹没在水中的angiocatheter<html> paratus的Microfil注射器。如果气泡据介绍，它往往可以被删除，只需继续Microfil灌注，直到泡沫已被推迟通过和冠状动脉血管。 血管网络可能没有填写完全的血管床的一部分，如果被封锁（图3B，箭头）。虽然肝素抑制血液凝块的形成，偶尔堵塞仍有可能出现由于不完整的肝素灌注前开始的程序，或由于其他未知因素。如果发生堵塞，还有的是，据我们所知，没有撞出的堵塞，完成血管填充的方法。如果太少，压力灌浆，Microfil将不能强迫所有的血管床和毛细血管网（图3C），也可以导致不完整的灌装。相反，压力太大，可能会导致毛细血管破裂，渗出到周围的组织Microfil（图3D）。 files/ftp_upload/3740/3740fig1.jpg"ALT ="图1"/&gt; 图1。 Microfil灌注计划概述 。 （一）主动脉和PVC削减约膜片的水平。 （二），升主动脉插管与angiocatheter。 （三）血管舒张缓冲区灌注通过压力灌注设备（图）驾驶的船只，而（四）主动脉弓离断三个主要分支结扎。 （五）通过的冠状动脉灌注，而4％PFA是两个前腔Cavas的结扎。 （六）使用注射器，Microfil通过的冠状动脉灌注，直到它被发现从PVC退出。 <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="图2"/> 图2。灌流装置。加入两个锥形瓶，每个充满要么舒张缓冲区或4％的煤灰，通过连接到他们的sidearms管加压。该系统是通过人工抽水加压灯泡，压力表连接到瓶，允许压力的监测和维护。小管，延长橡胶瓶塞通过上下将每个烧瓶中的液体。步枪和手枪泵的流体压力进入每个烧瓶出这些小管。管，然后在活塞只允许液体从一个烧瓶流在时间合并。 <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="图3"/> 图3。申请Microfilled心中 。 （一）充满良好的船只将很少有休息的Microfil（如有），及心脏组织将色彩的Microfil的颜色填充毛细管（星，并与C比较）。两个动脉（箭头 - 左前降支）和静脉（箭头 - 左冠状静脉）通过心脏表面可见。 （B）在microfil休息（星号），以及在一些血管堵塞的心脏，阻止完整的麦克风rofil渗透。封锁的船只保持红色（箭头），血液灌注过程中不会刷新。 （C）心脏血管不完全充满。请注意该组织一直没有采取的Microfil黄色，表明Microfil没有渗透到毛细血管。 （D）心脏毛细血管爆裂灌浆，造成Microfil泄漏到周围组织（箭头）。

<ol>
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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>

小鼠与制度的动物护理和使用华盛顿大学委员会批准，并按照由美国国立卫生研究院（NIH出版物，编号85-23出版实验动物护理和使用指南的方法处理， 1996年修订）。

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动脉血管的血管与不透X线材料填充的方法。该协议是专门设计，以确保所有的冠状血管，包括毛细血管，完整的馅，和随后的可视化。 为了确保完成所有的毛细血管充盈，充盈AGENT，Microfil，必须注入部分封闭的系统，将迫使较小的，更高的阻力血管内的血管网络的Microfil。要创建局部封闭灌装系统在我们的体内 ，我们的三个大，低阻力动脉从主动脉弓分支结扎。虽然这并不排除其他所有潜在的"泄漏"点，其余的船只（主要是肋间动脉）有足够小，任何压力，通过他们失去了不干预冠状动脉血管系统的完整的灌装。一旦的Microfil已灌注在心脏血管，心脏和血管组织的弹性性质的先天会挤压Microfil一些船只。为了防止这种情况的Microfil的损失，我们访问所有大型和退出点，即cavae两个腔，后腔静脉，主动脉，冠状动脉血管被完全灌注后结扎。在这种方式中，压力是m在心脏aintained，直到的Microfil聚合，允许Microfil符合正常生理血压下的血管结构。 另外，如果不需要可视化的毛细血管，它也可以只填写动脉或静脉血管床。 Microfil更多粘性版本可以混合使用高粘度的稀释剂（可从FlowTech）。更粘稠的灌流是无法渗透的毛细血管，因此允许可视化，如果从静脉侧灌注动脉，或仅脉。此外，该协议提出，在这里可以很容易地适应其他物种或者非成年小鼠。缩放的程序，适当搭配动物的大小，只需导管灌注管是适当的大小，以尽量减少动物的主动脉泄漏和防止拉伸或断裂。注入流体体积（即肝素，饱和KCl，Microfil）也必须适度规模。 然而，我们的协议是专门为μCT注射Microfil和成像设计，它可以很容易被改编为其他充填剂，无论是μCT分析，或其他前体内成像技术。 μCT兼容填料时，有几个选项透X线的染料，用于灌装和其他成像方法（如丙烯酸）通过学习血管的许多物质，可与无线电不透明的材料，如9领先色素或锇溶液注入， 3。无论使用的充填剂，μCT成像提供的结果可以改造成一个三维模型提供血管的测量以及填充6,7,14,19冠状动脉分枝格局的结构信息的优势。此外，由μCT成像保留周围组织，从而为额外1后扫描alyses。因此，填充和扫描的心中可作病理分析处理，及各种标记的染色部分可以μCT数据对齐相关动脉/静脉身份，平滑肌大衣的存在，或额外的病理指标。 其他常见的血管成像技术还需要血管充盈和我们的协议，可以很容易地适应，与任何其它充填剂灌注冠状动脉。扫描电子显微镜（SEM）需要填写的船只，然后从溶解软组织的进程中建立的血管称为腐蚀铸件的铸造。为了保持形状的船只没有对周围的软组织的支持，充填剂必须是强大的和非脆性:往往是丙烯酸树脂（如Mercox，巴特森的）3,20,21。虽然教统局局长提供μCT成像22，大大优于扫描决议，铸型ING程序破坏组织，防止任何额外的组织分析。 前体内的冠状动脉血管成像，光学投影层析成像（OPT）的另一种方法可以检测可见光或近可见光的光，从而允许除了色荧光信号检测碱性磷酸酶紫色沉淀析出等转换BCIP / NBT（5 -溴-4 -氯-3 -吲哚phosphate/4-nitro四氮唑蓝）23-25。船只的可视化，因此，可以实现通过与荧光物质（如PU4ii:聚氨酯树脂，或荧光素注入右旋糖酐26）灌装，或通过非填充改行荧光或方法，如整体安装免疫检测，组织化学显色沉淀（如BCIP / NBT）23。 OPT的成像可以实现略高于μCT（1微米左右）的决议;然而，灌装和immunodetection方法周围的软组织必须通过化学方法清除，这可能会破坏某些抗原的组织学分析后扫描。 也有不需要血管充盈或免疫检测的冠状动脉血管成像的几种方法，这样可以在体内进行。一个技术，对比度增强高分辨率超声（CEHRUS），利用造影剂充满气体的微泡。这些微泡注入到血液流允许可视化与实时流量测量血液流动的毛细血管水平，但它不提供的2,27-31成像船只3D视图。另一种方法，磁共振血管造影（MRA），也被用来32-34冠状动脉血管图像，并在MRA的最新进展，延长其成像能力，以获得实时的血流量测量35,36。虽然MRA能产生三维重建的VEMRA的决议ssels影像，目前仅限于100微米左右，因此无法确定小血管（毛细血管，小动脉和静脉）。 由于可以对活体动物进行，CEHRUS和MRA，他们多次和非侵入性监测血流量和血管的发展提供了优势。然而，MRA的相对较低的分辨率和3D功能CEHRUS缺乏排除冠状动脉网络作为一个整体的成像。因此，需要血管充盈剂或免疫检测前体内成像技术获取高分辨率三维信息的冠状动脉血管系统很重要，而在体内的技术，提供有价值的信息，有关功能的血管网络（即流数据，随着时间的推移） 。终点前体内成像在体内的时间过程分析相结合的冠状动脉血管的研究提供了一个功能强大的系统。 </p&gt;

<html><table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 1毫升注射器</td><td> <a href="http://www.bd.com/" target="_blank">碧迪</a> </td><td> BD-309602 </td><td></td></tr><tr><td> 1/2cc 29G半永久连接的针胰岛素注射器</td><td> <a href="http://www.bd.com/" target="_blank">碧迪</a> </td><td> BD-309306 </td><td></td></tr><tr><td> 2"×2"的纱布垫</td><td> Med101store.com </td><td> SKU的2208 </td><td></td></tr><tr><td> 24G¾"Angiocath静脉导管</td><td> <a href="http://www.bd.com/" target="_blank">碧迪</a> </td><td> BD-381112 </td><td></td></tr><tr><td> 26G半"表针</td><td>又长又"碧迪</td><td> BD-305111 </td><td></td></tr><tr><td>腺苷</td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">西格玛</a> </td><td> A9251 </td><td>舒张缓冲PBS 1G /升（与罂粟碱） </td></tr><tr><td>直角格雷夫钳</td><td> <a href="http://www.finescience.com/" target="_blank">罚款科学工具</a> </td><td> 11052-10 </td><td></td></tr><tr><td>棉签涂药:"非无菌</td><td> <a href="http://www.cardinal.com/" target="_blank">卡地纳健康</a> </td><td> C15055-006 </td><td></td></tr><tr><td>弯曲的手术剪刀</td><td> <a href="http://www.finescience.com/" target="_blank">罚款科学工具</a> </td><td> 14085-09 </td><td></td></tr><tr><td>解剖立体光源</td><td> <a href="http://www.nikon.com/" target="_blank">尼康</a> </td><td>不适用</td><td>不适用</td></tr><tr><td>解剖托盘，11.5 x 7.5英寸</td><td> <a href="http://www.coleparmer.com/" target="_blank">科尔-帕默</a> </td><td> YO-10915-12 </td><td>焦油充满牵制鼠标</td></tr><tr><td>精细弯钳</td><td> <a href="http://www.aesculapusa.com/" target="_blank">蛇牌</a> </td><td> FD281R </td><td>需要两个</td></tr><tr><td>肝素5000 U / ml的股票</td><td> <a href="http://www.apppharma.com/" target="_blank">APP的制药有限责任公司</a> </td><td> NDC的63323-047-10 </td><td> 1:100稀释水</td></tr><tr><td>氯化钾</td><td> <a href="http://www.fishersci.com/" target="_blank">费舍尔</a> </td><td> P217 </td><td>在水中的饱和溶液</td></tr><tr><td>氯胺酮（Ketaset），100毫克/毫升股票</td><td>道奇堡，欧弗兰帕克，堪萨斯州，美国</td><td> NDC 0856-2013-01 </td><td>为130毫克/千克体重，混合二甲苯胺噻嗪在0.9％s艾琳</td></tr><tr><td> microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">流技术</a> </td><td> MV-122（黄色）。其他颜色的选择也可以。 </td><td>重量1:1混合，固化剂量的10％。只是前准备，以确保它是很好的混合，注射和涡</td></tr><tr><td>非无菌缝合:6-0，编织丝绸</td><td> <a href="http://www.harvardapparatus.com/" target="_blank">哈佛仪器</a> </td><td> 723287 </td><td></td></tr><tr><td>罂粟碱</td><td> <a href="http://www.americanregent.com/" target="_blank">美国丽晶公司</a> </td><td> NDC 0517-4010-01 </td><td>在舒张缓冲PBS 4毫克/升（与腺苷） </td></tr><tr><td>多聚甲醛</td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">西格玛</a> </td><td> P6148 </td><td>准备为4％的解决方案</td></tr><tr><td>灌流装置</td><td></td><td></td><td>见图2 </td></tr><tr><td>弹簧剪刀</td><td> <a href="http://www.finescience.com/" target="_blank">罚款科学工具</a> </td><td> 15018-10 </td><td></td></tr><tr><td>甲苯噻嗪（Anased），20毫克/ GL股票</td><td> <a href="http://www.lloydlab.com/" target="_blank">劳埃德实验室</a> </td><td> NADA的＃139-236 </td><td>为8.8毫克/公斤体重，氯胺酮，0.9％生理盐水混合</td></tr></tbody></table>

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

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&lt;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。

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

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。

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

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

Nanyang Technological University

Research

JoVE Journal

Medicine

32.4K 次观看.  University of Washington. 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。

视频: 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.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

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

科研

医学

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

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.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

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.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

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.

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

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.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

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

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

小鼠脑海绵体畸形的诱导及显微 CT 成像

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

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.

非门控断层扫描的冠状动脉钙化识别

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.

小型动物正电子发射断层扫描/计算机断层扫描中的连续血液采样支持动脉输入函数的测量

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.

用于慢性病大型哺乳动物心脏模型对比增强显影显微计算机断层扫描的组织制备技术

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