我们要感谢气候变化框架公约实验动物设施为他们在这个项目的支持。我们感谢沃尔夫Laboratories公司为他们在分析血浆和组织中紫杉醇水平的援助。这项工作是由美国国立卫生研究院授予K01CA120091到EHB，并CA06927福克斯蔡斯癌症研究中心的支持。

该协议已获得的Fox Chase癌症中心实验动物管理和使用委员会和实验动物设施，并发现符合人道对待动物组织指导方针。 1.前期准备<ol><li>制备导管的:从一个短长度的聚乙烯管，改性，以形成变薄的，钝端（ 图3A）制备的导管。使多个导管提前和保存下去。 <ol><li>点燃本生灯，并调整建立了稳定的低火焰。持有油管靠近火焰软化聚乙烯。当管道开始熔化，慢慢拉开的两端以创建管道的薄壁部，大约0.25毫米外径。 </li><li>切坡口沿着薄款约为0.75公分。这样可以确保足够的管被固定在动脉，而无需创建一个超长的导管。 注:长的细端日Ë导管有一个倾向堵塞。超长年底还可能保持足够的液体以显著改变导管的液体体积容量。 </li><li>当适当加热，最终变得略显圆润，并扩大 - 通过快速穿过火焰生硬的坡口。尖钩小幅回升，这有助于锚管在插入到动脉。 </li><li>从那里开始瘦了点切管6.0厘米。这使得一个非常易于管理的导管，这是足够长的时间，通过线程退出，从颈部和持有和使用舒适的工作，但足够短，以防止鼠标啃上多余的管道，也不需要太多额外的输液量，以清除最初的生理盐水。 </li><li>准备约为0.2 ml肝素溶液的注射器，淋上用钝针。将针头插入导管（ 图3B）的广角端。填充导管，肝素，小心以确保在无气泡油管。在无菌区将肝素的注射器和导管一边。消毒导管通过γ照射，通过将导管（多个）的培养皿中，并暴露于γ-照射20戈瑞。如果您没有访问到的伽玛射线源，请与您的动物设施调查等方式消毒，如天然气或化学消毒。不要高压，聚乙烯，不能加热灭菌。 </li></ol></li><li>创建一个盐水引线（ 图3B）。 <ol><li>准备以约0.5毫升第二注射器无菌生理盐水，小心以确保无气泡线。 </li><li>切的第二管件的，大约为15厘米，并且滑动到修成平头的注射器针头。附上一个连接器端口的管的自由端。 </li><li>测试盐水的该流动通畅，通过引线顺利前进一个小体积的生理盐水。使用这种盐引导管插入后，检查过流该导管，并以冲洗血液的线。在无菌区预留生理盐水的注射器。 </li></ol></li><li>在手术之前，消毒设备通过高压灭菌，或者通过气体灭菌或玻璃珠灭菌。 </li><li>准备无菌手术区。 <ol><li>擦拭工作台，显微镜表面消毒剂，如70％乙醇或二氧化氯。盖台和显微镜的底座采用了干净的一次性吸水垫。 </li><li>由覆盖有两层清洁，吸收纸，牢固地附着有粘接带制备的外科板。 </li></ol></li><li>奠定了所有的手术耗材（如编目的材料清单），使它们容易获得。 <ol><li>切三种长度的无菌缝合，每个8厘米，并预留与其他物资。放置一个端口，这将是容易获得的（ 图3B）。 </li></ol></li></ol> 2.手术<ol><li>动物的准备<ol><li> Anesthetize老鼠暴露在2-3％异氟醚麻醉室连接到蒸发器的精度。从室撤回鼠标和鼠标的颈部/躯干上部刮毛，并在下面的右耳（现场导管出口处的）。负责兽医凡士林眼药膏眼睛，防止干涩，而在麻醉下。确保动物在手术准备通过使小鼠充分的时间在吸入室中制备（至少2分钟）之前，或者通过制备过程中通过鼻锥的手段施用异氟烷对小鼠不唤醒。返回鼠标麻醉室。 </li><li>当鼠标足够的惰性，转移到无菌手术区，将麻醉鼻锥在鼠标的鼻子和嘴，异氟醚转移到鼻锥流量。确认正确的麻醉捏掌钳;当鼠标显示无反应，请继续下一步。 </li><li>把鼠标在它的后面，与头部的面朝向研究者。保护耳朵，脱颖而出，爪子，后肢爪子外科板用胶带或其他限制装置，以保持鼠标的稳定。清洁用聚维酮碘和70％乙醇的切口区域。 </li></ol></li><li>颈动脉的隔离<ol><li>使1cm的纵向略切到颈部的中线的右侧。使用镊子分离脂肪和肌肉以暴露气管（ 图4A）。定位跑步平行于气管（ 图4B）的颈动脉。 </li><li>小心使用镊子分离筋膜覆盖在动脉（ 图4C）。轻轻从颈拉迷走神经一旁，并插入到钳子之间的空间。轻轻打开钳创建的筋膜间隙，然后小心地拉离动脉神经，从岔路口附近的喉部（前端）的动脉，同比增长（后部）尽可能（至少3mm）（ 图4D）。 </li><li>清除任何重新maining筋膜直至动脉是良好分离的（ 图4E）。加一滴生理盐水对手术区域偶尔组织保持湿润，从而减少脆，不易撕裂随机。 </li></ol></li><li>缝合位置和动脉准备的导管插入（ 图4，图5）。 <ol><li>使用镊子画动脉下丝缝合线。扎一个安全结到尽可能封闭动脉朝向前尽可能（ 图4F）。 </li><li>绘制动脉下第二个线程。配合可伸缩结暂时关闭掉动脉尽可能朝着后尽可能（ 图4F）。 </li><li>绘制动脉下三分之一线程。配合前两个缝线之间一个非常宽松的结，以用于快速固定后放置（ 图4G）的导管。 </li><li>用了一下70％的乙醇润湿他们持有的所有缝线出来的方式结束。 </li></ol></li><l我&gt;导管插入<ol><li>随着缝线，抢较低的结拉动脉稍拉紧。尼克上面的动脉，但非常接近，前部缝合（ 图4H）。要注意不要剪得太深，但检查的缝隙，以确保开口通畅。 </li><li>从注射器针头取下肝素充满导管，尽量避免在两端产生空气的大口袋。操纵导管在一个舒适的角度，斜角的位置，一般向下，并稍微向右侧（右撇子）。 </li><li>按住上缝合，以保持动脉剩余稍微拉紧，轻轻插入导管进入缝隙（ 图4I）。使用镊子举行前缝线和拉下来动脉导管以上（推过的导管可能会导致斜削端刺破动脉）。仔细释放导管和前缝合。 </li></ol></li><li>导管的固定和血流启动（图4J，图5B）。 <ol><li>通过拧紧中间缝线打结，靠近导管的入口进入动脉固定导管。使紧张的三重结，但一定不要拉那么紧，阻碍气流通过导管。通过与前缝线打结下来，入口下面的动脉进一步固定导管。 </li><li>附加的盐水引到导管的连接器插头的装置，再一次尝试，以避免引入气泡进入线。 </li><li>抓住后缝线的两端，轻轻拉动释放的结。机动缝合下动脉，在导管末端（不要删除线程）。血液应该流入导管;如果不存在血流量，轻轻摆动导管以试图去除收缩。 </li><li>当流量出现通畅，使用最后的线程（从后缝合），以配合额外的结，略高于中间缝合。 </li></ol></li><li>特导管mporary密封。冲洗血液的导管，然后使用止血钳导管靠近所述连接器插头的末端。卸下连接器，并将其与端口插头替换以密封导管的端部，然后取出止血。 </li><li>重新定位导管从颈背部退出。 <ol><li>在每个手钳，用一对钳子的时间来保持正下方的前部缝合的导管，并与其他中，按一个扭结到导管所以它很容易弯曲到侧面。重复创建第二个纠结。这使得导管的自由端以朝向鼠标的头部的背面拉出，而不强制导管的尖端转动横向对动脉壁。 </li><li>转动鼠标到其（左）侧，保持鼻锥体定位在嘴和鼻子，并清洗，用70％乙醇和聚维酮碘的切口区域。做一个小切口（约4m​​m）下方，右耳后面。 </li&gt; <li>使用镊子举行的皮肤瓣开放，而工作在皮肤上的生硬空洞探测过脸颊创建一个频道，向腔内的脖子。明智的做法是把，而不是试图腺体与皮肤之间去探测周围的唾液腺。使用镊子小心地免费为探针退出的空间。 </li><li>通过探头螺纹口塞/导管颈退出。不要拉太硬;可以肯定的导管未压碎或收缩血管或器官。 </li></ol></li><li>关闭和恢复。施用局部止痛剂（ 例如布比卡因）到肩部的切口，并覆盖有防水，外科胶带伤口。申请的第二片的粘合带，以进一步固定导管。 <ol><li>管理外用止痛胸部切口，并关闭创面用丝线或订书钉。 </li><li>从麻醉中取出小鼠，并允许动物在一个干净的恢复，加温加热垫的顶部空间（地点笼或在加热灯下），至少30分钟。 </li></ol></li></ol> 3.输液<ol><li>制备的5mg / ml的溶液紫杉醇/甲醇的等分试样。 <ol><li>测量50毫克的紫杉醇入15ml离心管中。加入10毫升无菌甲醇。帽筒。通过手工或在旋转摇动器在室温下旋转，直至粉末溶解。 </li><li>等份加入500μl的溶液至20小时，冷冻安全管，并储存在-20℃。 </li><li>解冻个别等分在室温下或在37℃的水浴中，立即输注之前。 </li></ol></li><li>准备输液泵（ 图6）。 <ol><li>切成大约40厘米聚乙烯管的长度长。附加一个钝针的一端，以及连接器端口到另一个。 </li><li>制定药物与一内径已知的注射器（最可编程的泵将所需要的注射器管的直径，以计算日的速度Ë泵臂）。连接针注射器和通过所述针和管装载药物。 </li><li>根据制造商的指示位于所述注射器进入泵内。灌泵，以使药物被顺利地流出的连接器插接件，并准备用于输液。 </li></ol></li><li>鼠标连接泵。 <ol><li>按住鼠标稳定，使用止血钳导管，靠近港口插头。除去塞子，并替换为附连到注射器和管接头。 注:血液可能开始通过管道回流。 </li><li>迅速地管理一个快速泵以清除导管的体积（通过观察6厘米管子的体积计算出的经验），然后立即切换到所需的输注速率。 </li></ol></li><li>继续紫杉醇注射了三个小时的课程。 <ol><li>监测管偶尔来检查是否有泄漏在路口，因为这往往是流动鼠标堵塞的迹象。观看鼠标预期的或意外的反应，输液（嗜睡或亢进，不适的迹象）。 </li><li>根据输液的长度和性质，鼠标可能无法进食或喝水，但一定要根据该机构的既定政策，提供获得食物和水。要知道，通过收集大量的血液脱水鼠标的潜力。 </li><li>继续保持温暖笼用加热垫或灯，除非鼠标似乎渴望远离热量。如果动物内的几个小时的手术后不施以安乐死，实施方案为外科手术后的治疗的动物，包括无菌的住房条件和治疗手术后疼痛。 </li><li>密切监视鼠标，特别是在最初的几个分钟，以确保它不会拉出，通过多动管路，或刺激，从管道（可能是一个贫穷的插入的符号）。如果鼠标没有过度活跃，不​​断的监测，环可能不是必需的，但检查小鼠常规地，以确保动物没有得到在油管纠结。安全带和绳索系统都可以买到，但它们的使用已经超出了协议的范围。 </li></ol></li><li>采集样品。 <ol><li>收集血样定期通过颌下或眶后出血（如果协议不使用乳腺肿瘤模型中，通过在同一时间作为颈动脉导管插入颈静脉导管考虑采集的血液）。要小心，不要拔输液管。如果通过眶后放血收集，轻轻麻醉小鼠用吸入麻醉剂（ 例如，甲氧氟烷），所以人们不必通过颈背抓住固定的小鼠。 </li><li>旋血液中hemocrit离心机以血细胞从血浆中分离出来。使用文件或钻石尖笔管的得分在脸相分离。破管只收集血浆，成小，冰柜​​安全管。店 -80℃下直至分析。 注意:如果一个hemocrit离心机不可用时，在高速传送血液样品放入微量离心管中，并在旋转的微型离心分离血细胞从血浆中分离出来。收集血浆成的第二管，并储存于-80℃。 </li><li>通过CO 2窒息安乐死鼠标。收集组织 - 从目的和闪冻于液氮中的器官（约20 50毫克）。储存在-80℃直至分析。 </li></ol></li></ol> 4.样品分析注:所有样品为这个协议是通过外部实验室用液相色谱-串联质谱法分析（LC - MS / MS），计算出的紫杉醇浓度谁如下: <ol><li>从样品中提取的紫杉醇。均化在0.1％乙酸的组织样品，在提取之前50％的甲醇。提取紫杉醇通过液/液萃取，用甲基叔丁基醚（MTBE）强化内部模拟（多西他赛）的标准。去除MTBE和干样品。重新悬浮于50％乙腈，0.1％乙酸溶液。 </li><li>准备校准标准。加紫杉醇的已知浓度到合适的C57BL / 6的矩阵，得到标准的最终范围（从1〜20,000纳克/毫升的血浆样品，在0.1至5000毫微克/毫升为组织样品）。提取标准，一式两份，用同样的方法，对所研究的样品的上方。测量中使用电喷雾离子化通过HPLC / MS / MS的样品紫杉醇峰。 </li><li>使用紫杉醇的面积比为内标计算出浓度。使用校准标准，配合曲线上建立一个标准曲线，插值研究样本。通过样本的同质化前的起始重量标准化研究样本的浓度。 </li></ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+Animal+Usage+by+Serial+Bleeding+of+Mice+for+Pharmacokinetic+Studies:+Application+of+Robotic+Sample+Preparation+and+Fast+Liquid+Chromatography+&#8211;+Mass+Spectrometry.">Reduction of Animal Usage by Serial Bleeding of Mice for Pharmacokinetic Studies: Application of Robotic Sample Preparation and Fast Liquid Chromatography &#8211; Mass Spectrometry.</a> Journal of Chromatography B: Biomedical Sciences and Applications. 754 (1), 245-251 (2001).</li><li>Berndt, K., Vogel, J., Buehler, C., Vogt, P., Born, W., Fuchs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+repeated+drug+infusion+into+the+femoral+artery+of+mice.">A new method for repeated drug infusion into the femoral artery of mice.</a> J. Am. Assoc. Lab Animal Sci. 51 (6), 825-831 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Squibb+&amp;+Sons,+L.L.C..">Squibb &amp; Sons, L.L.C..</a> Drug Information for TAXOL (Paclitaxel) Injection. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abraxis+BioScience+L.L.C.">Abraxis BioScience L.L.C.</a> Full Prescribing Information ABRAXANE for Injectable suspension (paclitaxel protein-bound particles for injectable suspension) (albumin-bound). , (2013).</li><li>Fergusson, G., Ethier, M., Zarrouki, B., Font&#233;s, G., Poitout, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Model+of+Chronic+Nutrient+Infusion+in+the+Rat.">A Model of Chronic Nutrient Infusion in the Rat.</a> J. Vis. Exp. (78), (2013).</li></ol>

作者什么都没有透露。

颈动脉灌注是在本研究中紫杉醇的药代动力学的显著技术。颈动脉灌注是一种方法在整个循环系统14快速分发药品。 3小时输液是一种更接近模拟药物临床管理，如紫杉烷类化合物比推注。该手术可以由单个个体可靠地进行，手术时间相对较短，并且成功率&gt; 75％。样品收集后，必须用适当的方法进行分析。我们用质谱测定血浆和组织样品中的紫杉醇浓度。为了进一步验证这项技术，我们...

药物输注通过颈动脉，可以通过优化的设备和技术，可靠和可重复地进行。该过程是不复杂的，但它确实需要精细的控制和对细节的关注。优异的护理和灵巧需要，分离出颈动脉并插入导管，其通常可以通过实践来获得。手术由经验丰富的技术人员不得超过一小时。手术成功后，鼠标应显示正常和健康的（虽然鼠标可反应的实际药物输注），以及药物（多个）可以在受​​控的，均匀连续的剂量给药。血液样本必须取自一个网站比颈动脉等;眼眶出血被证明易于收集和令人满意的药物浓度分析。 最佳尺寸和形状的导管是在执行一个成功的输液11制胜的法宝。我们发现导管可用commerciallÝ往往过大和/或太柔软，以允许方便地访问小鼠的颈动脉。它证明​​了优选从用于将鼠标连接到输注注射器的聚乙烯管材时尚导管。因此，所有的管道，连接器和针是一致的尺寸，这简化了输液组件的。使用这种技术，它是没有必要的导管的尖端推入动脉过去的地步，它仍然是可见的，并且血流量颈动脉未恢复，直到经过所述导管最初固定。这减少或具有导管的穿刺动脉的危害压出血流的高压。该导管的设计本文不包含一个"凸点"，以保持其位置，所以固定好导管用缝线和手术胶带是当务之急。 输注可以是优选的共同静脉推注，作为更好地模拟了临床给药药物，如紫杉烷类3，12，13，在这里所描述的技术最初被开发为允许注入到小鼠模型，其特征在于访问颈静脉或股静脉排除由乳腺肿瘤的生长和/或所述插入区的过度血管化。这种方法往往可能合适，即使在无肿瘤小鼠:虽然分离和插入导管颈稍微更具侵入性的，我们发现最好的颈静脉，因颈壁的倾向裂口导致多个失败插入和故障，完成在3小时的时间过程。 虽然这里示出的结果是从C57BL / 6J（内部饲养的）小鼠中，我们已经使用此技术成功地注入紫杉醇成几个品系的小鼠，包括FVB和混合菌株，跟随在小鼠模型转基因操纵的药代动力学下调细胞转运功能。血液和组织样品收集表明预期paclitax的水平埃尔，在颈静脉输注1后见到的水平的范围。这种技术可预期工作得同样好于其他小鼠模型，并与其它输注液。

<table><tbody><tr><td>Polyethylene tubing 0.024&amp;rdquo; OD X 0.011&amp;rdquo; ID&amp;nbsp;</td><td>Braintree Scientific, Inc.</td><td>PE10</td><td></td></tr><tr><td>3 Blunted needles (30 gauge)</td><td>Braintree Scientific, Inc.</td><td>NB-30</td><td></td></tr><tr><td>Stainless steel port plug (28 gauge)</td><td>Braintree Scientific, Inc.</td><td>PP-28</td><td>Slightly larger than PE tubing ID, to fit snugly and keep a tight seal.</td></tr><tr><td>2 Stainless steel connector plugs (30 gauge)</td><td>Braintree Scientific, Inc.</td><td>C-30</td><td></td></tr><tr><td>Three 1 cc syringes</td><td>Becton, Dickinson and Co.</td><td>309659</td><td></td></tr><tr><td>Sterile 0.9% Saline solution</td><td>Hospira</td><td>0409-7984-37</td><td></td></tr><tr><td>Cath-Loc HGS Heparin/Glycerol Solution&amp;nbsp;</td><td>Braintree Scientific, Inc.</td><td>HGS</td><td></td></tr><tr><td>Silk suture</td><td>Braintree Scientific, Inc.</td><td>SUT-S&amp;nbsp;113</td><td></td></tr><tr><td>Vanna Scissors (micro-scissors)</td><td>World Precision Instruments</td><td>14122</td><td>This model has a curved tip, but straight-tip scissors work as well.</td></tr><tr><td>Hartman Mesquito Hemostatic Forceps</td><td>World Precision Instruments</td><td>501705</td><td></td></tr><tr><td>Betadine Swabsticks</td><td>Perdue Products L.P.</td><td>BSWS1S</td><td></td></tr><tr><td>Bupivacaine</td><td>Hospira</td><td>0409-1160-01</td><td>May be replaced with Lidocaine, or similar local anesthesia.</td></tr><tr><td>Paclitaxel</td><td>LC Laboratories</td><td>P-9600</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>32213</td><td></td></tr><tr><td>Micro-Hematocrit Capillary Tubes, Heparinized</td><td>Fisher Scientific</td><td>22-362-566</td><td></td></tr><tr><td>Micro Capillary Tube Sealant&amp;nbsp;</td><td>Fisher Scientific</td><td>02-678</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Fox Chase Cancer Center, Laboratory Animal Facility in-house-bred</td><td></td><td></td></tr><tr><td>API 4000 Q-Trap mass spetrometer</td><td>Applied Biosystems</td><td></td><td></td></tr></tbody></table>

carotid artery infusions for pharmacokinetic and pharmacodynamic analysis of taxanes in mice

当提议使用一种药物，药物组合，或药物递送到一个新的系统中，一个必须评估药物的药代动力学研究中的模型。作为使用的小鼠模型是经常在临床前药物发现和药物开发1-8的一个重要步骤，有必要设计一种系统引入药到小鼠中的均匀，可重复的方式。理想情况下，该系统应允许血液样本的采集以规则的间隔在一组时间过程。测药物浓度通过质谱的能力，允许调查者跟着变化血浆药物水平随时间推移在个体小鼠1中，9，10。在本研究中，紫杉醇被引入的转基因小鼠作为连续动脉灌注在三个小时，而被同时取出的血样通过眼眶后出血，在设定的时间点。颈动脉输注是一个潜在的替代颈静脉输注，当因素如乳腺肿瘤或其他障碍物使颈输液不切实际的。使用这种技术，紫杉醇浓度在血浆和组织中达到相似的水平相比，颈静脉输注。在本文中，我们将展示如何成功地通过制备一种优化的导管，用于单个小鼠模型导尿颈动脉，然后展示如何插入并固定导管进入小鼠的颈动脉，螺纹的导管的端部通过所述背面鼠标的颈部，和钩鼠标的泵来输送药物涌入的受控速率。多个低容量眼眶出血允许血浆药物浓度随时间的分析。

<html>紫杉醇分布如下在15分钟的高速输注3小时，给药方案可预测的模式，随后是165分钟的低速输注。 图1示出颈静脉输注血浆紫杉醇浓度和颈动脉输注的比较。紫杉醇的浓度迅速下降，在第15分钟以下的初始高体积输注，然后在接下来的150分钟平稳。通过比较，在差的输注紫杉醇水平开始比较低，而且盘旋向上并在整个实验下来。这最有可能是由于在输液早期的?...

Watch this Scientific Journal Video about Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice at JoVE.com

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

该方法通过颈动脉递送稳定的药物溶液，以评估在小鼠模型中的新颖的药物的药代动力学的目标开发的。

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

carotid-artery-infusions-for-pharmacokinetic-pharmacodynamic-analysis

Research

Education

JoVE Core

JoVE Journal

Pharmacokinetics and Pharmacodynamics

Medicine

Unit 1

Pharmacokinetic Models

SCIENTISTS IN ACTION

22.9K 次观看.  Fox Chase Cancer Center. 该方法通过颈动脉递送稳定的药物溶液，以评估在小鼠模型中的新颖的药物的药代动力学的目标开发的。 

视频: Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

科研

医学

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

颈动脉肿瘤细胞注射，产生脑转移瘤小鼠模型

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

癌症研究

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

通过颈内动脉注射癌细胞模拟脑转移

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

颅内插管植入小鼠连续局部区域嵌合抗原受体（CAR）T细胞输注

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

<meta charset="utf8"></head><body>小鼠模型中注射后的颈总动脉修复

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

<meta charset="utf8"></head><body>作者聚焦:通过溶瘤病毒和 MicroRNA 的动脉内递送策略推进胶质母细胞瘤治疗

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

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一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

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通过颈内动脉注射癌细胞模拟脑转移

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化疗引起的血管毒性 - 实时

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统