作者要感谢安德鲁博士王，博士和“卡特里娜”国王的插图的贡献。国立卫生研究院授予：R00HL087927。

1。外科手术开始之前 注：开始前的任何动物的程序，确保您获得适当的权限，通过您的机构/组织。至于与所有的生存手术，确保手术是在无菌条件下进行适当的止痛药和抗生素的需要，是取得成功的结果。 <ol><li>开始前导尿过程评估导管，用无菌生理盐水冲洗，以确保他们的专利。 </li><li>麻醉大鼠。 </li><li>准备手术过程中的老鼠。 <ol class="lalpha"><li>剃须从手术区域，其中包括颈后（肩胛骨之间）和内部腿部地区的皮毛。 </li><li>分别使用优碘和70％乙醇擦洗，擦洗剃光手术开始在中心和一个圆形扫向外地区。重复每个地区3次，最后用70％乙醇清洗与整理。 </li><li>放置在无菌的表面和地方无菌手术区悬垂动物。 （新闻- N -密封的使用是一种理想的手术的悬垂性，使研究者监控整个过程中的动物。） </li></ol></li><li>确保手术过程所有的手术工具已经消毒。 </li></ol> 2。导管置入手术区的准备工作<ol><li>大鼠铺设容易发生它的肚子上，约一个半英寸（12毫米）的水平切口颈部背部肩胛骨用剪刀或手术刀，然后钝性解剖皮下的“口袋”的水平上回约四分之一大小。这将作为一个区域用来放置少量的油管，将动物的生长和/或运动补偿，即导管不拉和从动脉从而删除，这是地方ð英寸（或者这一步，可以立即执行之前，隧道导管。） </li><li>其背面（仰卧位）放置到大鼠和后腿自然的角度，在腹股沟区[近似半英寸（12毫米）切口沿切口。 </li><li>钝性解剖单独的结缔组织（图1）（可以使用钝尖的剪刀，止血，棉签等）（通常在45度角钝尖剪刀和/或棉签，这确保了更容易的本地化感兴趣的区域，直到股动脉和静脉）暴露。 <ol class="lalpha"><li>静脉色暗红，动脉比静脉更清晰和明亮。运行的神经沿动脉音发白。 </li></ol></li><li>钝性解剖的腿部地区沿腿部内侧的皮肤下面一个小的开放的地区（即口袋，约四分之一大小的一小部分的cathete安置）R（再考虑到动物运动和动物如果长期放置增长）。 </li><li>放入切口区的拉钩，使您可以完全查看动脉和静脉。 </li><li>使用精尖镊子轻轻分开沿着股动脉，离动脉和静脉的神经（颜色发白）。 （图2）注意不要切断或损伤神经。 </li><li>单独作为一个单元的动脉和静脉，试图揭露约¼英寸（5-7毫米）的动脉/静脉的长度部分。 </li><li>重复的过程，在分离的动脉，静脉。保持你的手术器械（即精尖钳）垂直的船只和独立并行的船只。这将有助于避免流泪，刺痛或损坏的船只。通过精尖镊子轻轻放在从下面缓缓开口钳和重复此之间的动脉和静脉，你会慢慢分开的船只。注意：如果你撕裂或观察一些出血使用无菌棉签和/或2 × 2纱布，放置在该地区的压力，直到出血停止，然后继续手术。 </li></ol> 3。隧道导管<ol><li>在切口放置无菌生理盐水浸泡2 × 2纱布，并打开了动物它的肚子。 </li><li>以前和引导下来回的臀部的皮下钳[确保止血提示指出，（对脊柱不回的切口放入罗切斯特Pean钳（长直钳） ），以避免伤及脊髓]。大约在打开髋部地区对在腿区域的切口止血钳尖端和推动尖端的止血准备腿部切口。 </li><li>轻轻抓住导管钳（而不是将插入到动脉/静脉完）年底，轻轻一拉，作出腔导管并最终出颈部切口。 </li><li>将适当的钝尖的注射器20 U / ml肝素/生理盐水填充各自的导管年底和填补与肝素/生理盐水的导管（确保没有气泡）。再次检查导管专利和无损伤导管。将注射器连接导管线年底，以确保没有空气进入导管线和再次转入大鼠背部。 </li></ol> 4。股静脉和动脉插入导管（图3） <ol><li>股静脉下放置一个折叠一块无菌4.0丝/线程，然后削减丝绸折叠结束。现在有两件丝绸下静脉。或者，单独下静脉放置2件4.0丝绸。 </li><li>你下静脉放置，丝绸一块对腿（末端）和其他对身体分开。 T宽松结扎即最接近身体的一侧，然后用小止血，把握丝绸和拉教，但不要拧紧结。拉丝绸等，尽可能对腿（末端），并配合成三结，把握小止血的丝绸和拉教。这种方法将允许静脉充满血液，使其更容易使切口插入导管（步骤4）的要求。 </li><li>放置1-2上滴利多卡因静脉。 </li><li>使用的万纳微解剖剪刀，做一个小切口静脉约¼通过，并在45度角。 </li><li>放入切口，用另一双钳细尖镊子（45钳）;仔细饲料中的静脉导管。轻轻打开放入静脉，因为这将允许研究者轻轻置于钳和静脉的静脉导管钳。 </li><li>当完全插入导管（欧普罗X. 6-7厘米）（导管时，一个标记是放置导管，以协助确定完全插入导管时，外科医生）（这个地方在腹部下腔静脉的静脉导管），拧紧前周围静脉结扎和导管，追平了三重结（确保它不阻断静脉）。用丝线缝合（后壁结扎）附近的腿再次安全的导管（三结），并确保安置。慢慢拉回注射器，直到有一点点的血液在导管中，这有助于确保缝合海里，不会太紧，导管功能可见。检查后，按下柱塞，直至血液不再是可见导管。 注意：它是可能的2导管插入股静脉，如果需要的话。 </li><li>重复步骤9-13放置股动脉导管有以下例外： <ol class="lalpha"><li>铁腿（后）的丝绸最近三重结和拉头GHT前前导管置入的切口搭售身体（前）附近的松散结扎。这将允许动脉，以填补与血液更容易削减。确保教之前削减动脉闭塞动脉近端缝合拉。切时，这将避免失血。 </li><li>插入动脉导管从股动脉约5厘米（这个地方在腹主动脉导管）。 </li><li>确保导管缝合时，确保不缝合过紧夹闭导管。 </li></ol></li></ol> 5。手术总结<ol><li>做一个毛钱季度导管和腿部内侧的地方（循环应符合钝性解剖早期的区域）的大小循环。后放置两个导管，安全与1-2 5.0到肌肉层的手术缝合针。 </li><li> 4.0非连续sutur Ethilon关闭切口ES。 </li><li>转入其胃大鼠，并在一季度和解剖在后面的口袋大小的导管的另一循环。关闭与缝合切口。 </li><li>使用vetbond下降，争取在后面的导管。 </li><li>背部切口附近的填充止血钳的导管和注射器从两端删除。 </li><li>适合系绳式外套的大鼠，第导管（保持肝素锁），并取出填充止血。 </li></ol> - 导管生理盐水溶液与20 U / ml肝素/生理盐水取代长期维护。 6。导管的维护（无菌手套应戴在程序） <ol><li>填充钳钳导管。 </li><li>取下导管插头。 </li><li>锁定导管的解决方案将一个钝尖注射器。 </li><li>松开镊子。 </li><li>填充导管锁解决方案（体积预先DETErmined - 通常是0.3毫升）。 </li><li>同时冲洗，以防止任何的血液回流到导管尖端，并取下注射器，导管钳的。 </li><li>更换导管堵塞。 </li><li>松开镊子，轻轻一推插头，以确保没有血液在导管尖端轻微。 </li></ol> 7。采血（无菌手套应戴在程序） 填充钳钳导管。 <ol><li>取下导管插头。 </li><li>慢慢地退出锁解决方案，采用尖钝的注射器和丢弃。 </li><li>将采样注射器，导管，慢慢撤回样品。 </li><li>锁解决方案填充钳钳将导管和注射器到导管，重新填写导管与锁定的解决方案。 </li><li>夹紧导管，而再填充导管的解决方案。 </li><li>重新插入导管堵塞。 </li><li>取出填充镊子轻轻推吨<html>他进一步轻微堵塞。 </li></ol> 8。药物输注<ol><li>填充钳钳导管。 </li><li>取下导管插头。 </li><li>慢慢地退出锁解决方案，采用尖钝的注射器和丢弃。 </li><li>导管将药物填充注射器注入到动物的实质。 <ul><li>可以使用3路阀，以及一个中间，如果需要多次注射，因此需要到动物少输液。 </li><li>还可以附加一个恒定的输液注射泵用连续输液的无菌过滤。 </li></ul></li><li>钳填充钳和锁定的解决方案上导管和注射器重新填写导管锁解决方案。 </li><li>夹紧导管，而再填充导管的解决方案。 </li><li>重新插入导管堵塞。 </li><li>取出填充镊子，轻轻一推进一步轻微堵塞。 </li></ol>E“&gt; 9。血压和心率采样<ol><li>填充钳钳动脉导管，取下导管堵塞。 </li><li>将压力传感器的动脉线。 </li><li>按照血压集合使用软件的制造指示。 </li><li>血压监测期结束，使用填充镊子夹住导管，断开传感器的。 </li><li>锁溶液冲洗导管和更换导管堵塞，如上所述。 </li></ol> 10。代表性的成果一个有代表性的血压测量是从有意识的自由活动的动物，并在图4。进入股静脉行，血压升高，去氧肾上腺素（3微克/公斤，四），一个α1肾上腺素受体激动剂，管理，同时测量血压从股动脉行。酚妥拉明（4毫克/千克，IV），非选择性α-肾上腺素受体拮抗剂，当时管理，降低血压。 <img alt="图1" src="/files/ftp_upload/3496/3496fig1.jpg" /> 图1。钝性分离组织。随着大鼠在他的背上，你将有后腿的角度大约一个半英寸（12毫米）切口，用钝的剪刀或止血，钝性解剖的结缔组织，暴露股动脉和静脉。 <img alt="图2" src="/files/ftp_upload/3496/3496fig2.jpg" /> 图2分离股动脉，静脉和结缔组织。 <img alt="图3" src="/files/ftp_upload/3496/3496fig3.jpg" /> 图3。导管的位置。使用万纳微解剖剪刀放置一个小切口约¼的方式通过船只在45度角（顶部）和细尖镊子放入第船只（中）Ë切口，并使用另一双钳送入导管。最后，缝合完成导管的位置后，导管到位（下）。 <img alt="图4" src="/files/ftp_upload/3496/3496fig4.jpg" /> 图4。代表性的血压测量，从有意识的自由活动的动物。

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动脉和静脉插管在历史上被用于急性和慢性监测血压，血液样本，并提供物质在大鼠实验动物模型 1-4 。这些手术的检测仪器的一个主要好处是，它允许监控程序，包括血液样本，药品监督管理和血压监测，要进行最少的干扰和/ 或动物的压力1。许多研究者的书面协议，并有具体的方法，他们的实验室进行外科手术 5-8 。视频和插图，证明我们的实验室已经发现关于股动脉和静脉插管过程中取得成功。 大鼠在实验室中常用的多种科学的研究由于其体积小，方便处理。有几个地点是其中的一种慢性导管可以放置在一个动物，包括颈内静脉，腹主动脉，颈动脉和股动脉，仅举几例。大鼠股骨的位置慢性导管检查结果和导管通畅的长度增加了术前动物体重比其他导管位置 9最快的恢复。 许多研究者使用留置导管对急性以及慢性血液抽样1-5,11-13。在许多研究中多个血液提请需要一种动物和外部插管/导尿是一个方法，由于它的非创伤性的性质是有利的;此外，它可以做到，而动物是有意识的，因而是不麻醉药的影响有限的动物，也可以自由移动的1,10。最好的方法获得的血液样本，并测量在啮齿类动物的应激激素一直争论 6,13 。对于药代动力学studiES，研究动物插管允许以最少的克制的反复血液样本时使用一个长期植入的导管系统。此外，有研究表明在自由活动大鼠颈静脉导管相比，动物被处理（30秒）或限制（5分钟）14血浆中肾上腺素，去甲肾上腺素和多巴胺的减少基底浓度。辨别应力增加一个额外的方法是通过测量血浆皮质酮水平。此前有人建议，甚至三到四个手术的恢复时间天15在慢性空心大鼠血浆皮质酮水平升高。然而，在最近的改进方法，已确定有 16 uncannulated大鼠基线空心大鼠颈静脉血浆皮质酮水平相比没有差异。此外，皮质酮分析方法高效液相色谱法还透露，corticoster之一应激水平升高;然而，稳定后，颈静脉导管插入术16。 额外使用的慢性导尿是在啮齿类动物的血压和心率的测量。有多种方法，是用来测量在大鼠血压和心脏率，这些包括非侵入性的尾套方法，无线电遥测程序和直接留置导尿管。每种方法都有其优点和缺点，这是其他出版物中详细描述。留置充满液体的导管可以被植入到大鼠内的多个动脉。股动脉，但这项措施可以使用一条动脉。对于血压/心率措施，导管末端连接到一个经过校准的压力传感器。该导管可以被安置在一个受保护的弹簧连接到一个旋转，允许自由流动的动物，或连接到一个按钮surgically植入动物。留置导尿管的优势，最大限度地减少对动物17,18的长期紧张。此外，材料便宜，校准压力的措施和持续的长期措施很容易 ，可众多19周的压力比较低的情况下取得的。我们将是失职，如果我们没有提到，这项技术也有缺点，包括的，它是外科微创技术，维护的导管必须保持通畅，损害动脉导管植入仅举几例的感染潜力。 对于慢性血压长期植入动脉导管直接录音措施在技术上具有挑战性的，但更准确，更可以做到不扰民的动物不断。尾袖带测量精确度较差，但他们并不需要手术治疗，也可重复编辑。尾袖方法确实需要处理以及动物的尾巴血管扩张，以方便检测的脉冲20加热。处理和热应激可影响血压测量，因此不能提供真正准确的措施。此外，非直接的尾巴袖套法不允许轻松的同时进行血液抽样或药品监督管理局。 一个额外的直接测量方法，通过它可以实现用遥测方法。遥测血压（和其他措施）的高品质的录音可以连续长时间意识的动物自由活动的时间，不加节制或麻醉剂18。然而，遥测设备非常昂贵，而理想。当比较到遥测，导尿的好处包括：“设置”下降和运营成本，能够随时管理药品和容易清醒自由活动的动物的血液样本。该administrat离子药物和物质，并从取得的研究动物血液样本，可以做微创令人不安的动物，从而减少动物的压力，并允许一个更准确的测量。

<html><table border="1"><tbody><tr><td> 名称 </td><td> 类型 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>动脉导管</td><td>耗材</td><td>世输液</td><td> RFA - 01 </td></tr><tr><td>静脉导管</td><td>耗材</td><td>科学品公司</td><td> BB518 - 20 BB31785 - V / 2 </td></tr><tr><td> 27克1 / 2“钝针</td><td>设备</td><td>集成点胶解决方案公司</td><td> 9991113 </td></tr><tr><td> 23克1 / 2“钝针</td><td>设备</td><td>集成点胶解决方案公司</td><td> 9991109 </td></tr><tr><td> <a href="http://www.adinstruments.com/products/hardware/research/product/PL3508/" target="_blank">PowerLab/8SP</a> </td><td>设备</td><td> <a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a> </td><td>w.adinstruments.com/products/hardware/research/product/PL3508 /“目标=”_blank“&gt; ML765 </td></tr><tr><td> <a href="http://www.adinstruments.com/products/hardware/research/product/FE224/" target="_blank">四桥</a> </td><td>设备</td><td> <a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a> </td><td> <a href="http://www.adinstruments.com/products/hardware/research/product/FE224/" target="_blank">ML118</a> </td></tr><tr><td> <a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 7.2</a> </td><td>软件</td><td> <a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a> </td><td></td></tr><tr><td> <a href="http://www.adinstruments.com/products/hardware/research/product/MLT844/" target="_blank">BP传感器与活塞电缆</a> </td><td>设备</td><td> <a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a></td><td> <a href="http://www.adinstruments.com/products/hardware/research/product/MLT844/" target="_blank">MLT844</a> </td></tr><tr><td>单信道旋转</td><td>设备</td><td> Instech </td><td> 375/22PS </td></tr><tr><td>单轴制衡旋转安装</td><td>设备</td><td> Instech </td><td> CM375BP </td></tr><tr><td> Covance公司输液线束</td><td>设备</td><td> Instech </td><td> CIH105 </td></tr><tr><td>台面异氟醚麻醉组</td><td>设备</td><td>保得国际公司</td><td> 61020 </td></tr><tr><td>鼠患的麻醉用面具</td><td>设备</td><td>保得国际公司</td><td> RAM - 02 </td></tr><tr><td>啮齿动物感应室</td><td>设备</td><td>保得国际公司</td><td> RIC - 01 </td></tr><tr><td>麻醉气体滤筒</td><td>设备</td><td>保得国际人，公司</td><td> 80120 </td></tr><tr><td>桌面层流罩</td><td>设备</td><td>哨兵航空系统公司</td><td> SS - 200 - WSL </td></tr><tr><td>奥斯特金A5 </td><td>设备</td><td>奥斯特专业产品</td><td> 78005-140 </td></tr><tr><td>光纤照明与双鹅颈指南</td><td>设备</td><td> LW科学公司</td><td>馆际互借- 1502 - DGG1 </td></tr><tr><td> Deltaphase恒温垫</td><td>设备</td><td>布伦特里科学</td><td> 39DP </td></tr><tr><td>棉尖模具</td><td>耗材</td><td>梭伦制造公司</td><td> 36200 </td></tr><tr><td>纱布垫2“X2” </td><td>耗材</td><td>肯德尔Curity </td><td> 2146 </td></tr><tr><td> 5-0尼龙线</td><td>耗材</td><td>爱惜康</td><td> 661G </td></tr><tr><td> 4-0丝绸缝合</td><td>耗材</td><td> DEKnatel </td><td> 8 - S，136075-0208 </td></tr><tr><td>组织粘合剂</td><td>耗材</td><td> 3M </td><td> 1469SB </td></tr><tr><td>分裂和固定钳</td><td>设备</td><td>乔治Tiemann公司</td><td> 160-55 </td></tr><tr><td>学生组织钳</td><td>设备</td><td>精细的科学工具，公司</td><td> 91121-12 </td></tr><tr><td>微钳/蕨菜钳</td><td>设备</td><td>乔治Tiemann公司</td><td> 10-1942 </td></tr><tr><td>超细格雷夫钳</td><td>设备</td><td>精细的科学工具，公司</td><td> 11152-10 </td></tr><tr><td>奥尔森的Hegar持针器</td><td>设备</td><td>精细的科学工具，公司</td><td> 12002-12 </td></tr><tr><td>学生霍尔斯特德蚊子止血</td><td>设备</td><td>精细的科学工具，公司</td><td> 91308-12 </td></tr><tr><td>罗切斯特Pean队p </td><td>设备</td><td>生物医学研究仪器公司</td><td> 31-1640 </td></tr><tr><td>学生手术剪刀</td><td>设备</td><td>精细的科学工具，公司</td><td> 91402-12 </td></tr><tr><td>杜蒙钳</td><td>设备</td><td>精细的科学工具，公司</td><td> 11251-35 </td></tr><tr><td>微解剖剪刀</td><td>设备</td><td>乔治Tiemann公司</td><td> 160-210 </td></tr><tr><td>斜视剪刀</td><td>设备</td><td>精细的科学工具，公司</td><td> 14075-11 </td></tr><tr><td>哈特曼止血</td><td>设备</td><td>精细的科学工具，公司</td><td> 13003-10 </td></tr><tr><td>组织剪刀</td><td>设备</td><td>乔治Tiemann公司</td><td> 160-150 </td></tr><tr><td>牵引器</td><td>设备</td><td>定做</td><td></td></tr></tbody></table>

femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements

在多个研究领域，获得在实验室研究中的循环系统是必要的。药理研究大鼠长期植入导管允许研究员，有效地和人道管理的物质，进行反复血液样本，并在有意识的直接测量血压和心率助攻。一旦被植入导管的长期采样是可能的。通畅，导管的使用寿命取决于多种因素，包括使用锁的解决方案，冲洗疗法和导管材料。此影片将展示大鼠股动脉和静脉导管插入术的方法。此外，影片将展示使用的股静脉和血液样本，药品管理和使用中有意识地自由移动大鼠血压和心率的测量动脉导管动脉导管。系绳和线束连接到一个旋转系统将使动物是何使用，并已采取样品，以最小的中断动物研究员。为了保持导管通畅，小心的导管日常维护需要使用锁解决方案（100 U / ml的肝素生理盐水），机地面先端钝的注射器针头和注射器过滤器的使用，以尽量减少潜在的污染。小心无菌外科技术，正确的导管材料和细致的导管维护技术，有可能持续很长一段时间（数周）专利导管和健康的动物。

Watch this Scientific Journal Video about Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements at JoVE.com

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

在大鼠血管慢性导尿往往需要物质的管理，获得过一段时间，或直接意识的血压测量血液样本。自觉动物血压的大鼠和相应的测量股动脉插管将被证明。

股动脉和静脉导管采血，药品监督管理局和意识的血压和心率测量

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

femoral-arterial-venous-catheterization-for-blood-sampling-drug

Research

JoVE Journal

Medicine

123.9K 次观看.  Michigan State University. 在大鼠血管慢性导尿往往需要物质的管理，获得过一段时间，或直接意识的血压测量血液样本。自觉动物血压的大鼠和相应的测量股动脉插管将被证明。

视频: Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

颈动脉和颈静脉导管在有意识大鼠模型进行血流动力学措施，输液和血液采样

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

科研

医学

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

根尖切除小鼠模型来研究早期哺乳动物心脏再生

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

分流手术，右心导管，血管形态计量学在大鼠模型中的流量引起的肺动脉高压

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板输血中微粒的常规筛选方法

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

吸收性微活检取样和 rna 提取用于微创同时进行血液和皮肤分析

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

大鼠同时心电图记录及侵入性血压测量

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...