这项研究是由加拿大健康研究和基因组Canada.JS研究院的支持下，拥有来自阿尔伯塔省文物Foundationfor医学研究，心脏及中风基金会，加拿大糖尿病协会的Canadaand的薪金支持奖。特别感谢博士大卫沃瑟曼和宾格尔布雷西此过程希勒实验室教学。

答:手术导管，动脉和静脉循环第1部分:动脉和静脉导管的制备<ol><li>切15厘米PE - 50（内部直径0.58毫米（0.023"）x外径0.965毫米（0.038"）。硅胶管切1毫米节（0.76毫米（0.030"）内部直径x1.65毫米（0.065"）外径），使用限制珠。限制珠防止拉出导管，一次到位的老鼠。 </li><li>微解剖钳插入管腔的限制珠的技巧和镊子轻轻托起的秘诀除了伸展扩大开放。使用另一微解剖钳，滑入管腔限制珠硅橡胶管。具有较强的粘合胶（乐泰超级胶）安全。珠必须平躺导管周围。允许导管干（24小时）来完成。 </li><li>对于一个300克重的老鼠，动脉行〜的限制珠颈行〜3.2厘米2.7厘米。从导管的制约长度是由0.5厘米调整为每100克体重的增加。不要船只在插入导管，因为这可能穿刺锥的边缘。 </li><li>立即在手术前，填写与肝素生理盐水（10U/ml）线，密封两端用不锈钢管堵塞和乙醇（70％）放置在消毒。空气干燥前插入。 </li></ol>第2部分:手术准备<ol><li>由卡尔加里动物保健大学和程序批准使用委员会和theCanadian协会遵守实验动物科学实验的准则。下面列出的程序进行了成年，雄性Sprague - Dawley大鼠（约300克）。所有程序下进行异氟醚，虽然也可以使用注射麻醉剂。所有的手术程序进行，确保无菌操作技术。外科设备，烧杯和肝素生理盐水高压灭菌消毒。从供应商购买的消毒，手术手套，注射器和棉倾斜涂药。 </li><li>称量大鼠，​​并记录结果。在手术后的监测动物的重量将是重要的的。应使用重拾重量只有到手术前水平的动物实验。在麻醉中麻醉大鼠（3％异氟醚）。保持在手术使用鼻锥的大鼠在1〜2％异氟醚。在消毒领域，促进无菌手术必须进行。 </li><li>准备消除手术部位的头发（颈部和肩胛骨）的动物。可以用一个小理发器或化学脱毛。从那里进行手术是在一个地区单独执行此过程。 </li><li>安全动物手术台。确保通过检查脚/眼反射的存在是完全麻醉的动物。准备适当的皮肤消毒剂（70％的乙醇，70％的乙醇优碘擦洗）在手术部位。 </li></ol>第3部分:手术<ol><li>小型垂直正中切口1厘米优于胸骨（拿起皮肤，纵向沿中轴与钳和剪刀或手术刀切割）。 </li><li>使用微解剖钳暴露左侧胸锁乳突肌肌钝性解剖。反映这种肌肉暴露左侧颈内动脉约1厘米。使用微解剖钳下颈内动脉动脉举行的地方。轻轻挑逗，从颈动脉结缔组织。重要的是从动脉的迷走神经分离不破坏动脉或神经。孤立的动脉，然后用4-0丝线缝合（这将是用来操纵在手术过程中的颈动脉）结扎头侧结束。注:70％的乙醇，使用前应消毒4-0缝线。 </li><li>带有锯齿的船只，微解剖钳钳。 21号VENOJECT多重采样露儿适配器的结扎穿刺结束。取出不锈钢管堵塞，小心地插入导管承认到动脉无菌导管导引援助。确保导管钳安全，部分放开微观解剖钳，夹闭动脉，并继续插入导管珠。此时，导管尖端应在主动脉弓。小心不要释放导管，血管的压力将迫使它。 </li><li>铁两个4-0缝合牢固下面珠和一个以上，并确认导管将样品。 10U/ml肝素生理盐水冲洗行。重新插入导管用不锈钢管堵塞的外部结束。 </li><li>使用相同的切口，钝性解剖暴露右侧颈外静脉。仔细分离，并用丝线缝合结扎头侧结束。静脉穿刺21号VENOJECT多重采样露儿适配器。取出不锈钢管堵塞，插入导管用无菌彗星atheter介绍人珠和配合下面的两个珠缝线。领带上面珠第三缝合，并确认它的样品。与静脉行10U/mL肝素生理盐水冲洗。不锈钢管插头重新插上外部导管末端。 </li><li>隧道14号钝针下的皮肤，使切口在肩胛骨之间的背部。穿过针的导管exteriorize大鼠背面。大约4厘米行是可见的。切的聚乙烯的S - 50 - HL医用导管0.5厘米，周围放置的exteriorized导管和安全需要（蓝色静脉，动脉红色）的录像带。复试的导管，以确保通畅，冲洗和填写与150U/ml肝素生理盐水，以防止凝血。 </li><li>关闭所有的切口，用3-0丝线缝合。放置鼠易发生，在预热，干净笼笼底的食物。 </li></ol> B:术后护理第1部分:术后护理和监测<ol><li>一旦老鼠重新获得充分的日间能力和警觉性大鼠返回畜舍。 </li><li>允许大鼠为3-5天恢复。 </li><li>每日监测感染，疼痛和体重的变化。如果放电观察切口网站，一般嗜睡和/或疼痛，感染可能是关注。疼痛是驼背的姿势，竖起背毛皮及日间和/或饮食行为的情况下表示。然而，可能会经历的减肥手术后手术后3天立即重量应稳定和/或增加在术前体重的10％在5天之内。严重的体重减轻可能表明感染，中风和/或疼痛。 </li><li>复试导管每天以确保通畅。 </li></ol>第2部分:保持导管通畅<ol><li>术后恢复期间的每一天，150U/ml肝素生理盐水填充1毫升滑提示注射器和第22号钝针。 23规格的不锈钢管耦合器的另一端插入到PE - 50管20厘米钝针。 </li><li>取下放置管耦合器的一端高于其余1ml注射器，推行的气泡，气泡。 </li><li>钳断动脉线从老鼠与止血略低于插件外部。 </li><li>使用止血第二对拆下的钢导管堵塞。 </li><li>插入动脉线连接到注射器管耦合，并释放止血，阻断外部的动脉导管。 </li><li>动脉导管的血液吸进注射器。如果导管不容易画，它可能需要轻轻推少量的清洗液通过导管打跑导管尖端情况下，它是对血管壁楔形。导管已完全清除，当血液中达到注射器。 </li><li> PE - 50管接近钳1ml注射器，注射器和处置。更换一个新的注射器充满新鲜150U/ml肝素生理盐水。松开油管，并举行新的注射器直立，手指轻弹与新的注射器逐出任何可能的新的气泡向上远离行。注意:必须确保气泡可导致中风。 </li><li>直到导管行是明确的和无血，注入150U/ml heparinzed生理盐水。 </li><li>从大鼠外部略低于管耦合器止血钳断动脉。从动脉导管行取出注射器连接器，并用不锈钢管堵塞替换。 </li><li>松开止血，阻断外部的动脉导管。 </li><li>重复静脉行。然而，由于静脉压力低采样往往是不可能的。如果150U/ml heparinzed生理盐水注入可以以最小的阻力很可能定位静脉导管。 </li></ol> C:高胰岛素 - 正常血糖钳第1部分:钳设置和准备<ol><li>称量大鼠和记录重量。这将需要确定胰岛素和葡萄糖输注率。快速的大鼠实验前至少5H。这将确保动物在餐后状态和食物来源的葡萄糖的贡献最小。放置在一个小容器/腾笼换床上用品，限制过度运动大鼠。动物应该能够扭转和新郎自由。 </li><li>设置如图1所示的线条和输液泵。 P50连接管长度如下:Y型连接器是注射器8厘米，对大鼠的Y型连接器是15厘米，动脉行20厘米，静脉行是在长10cm。删除不锈钢管插头，并刷新颈静脉和动脉线，以确保输液和采样10U/ml肝素生理盐水分别。 </li><li>确定胰岛素<html>所需体积。这将取决于对大鼠胰岛素水平和重量。在这里，4mU/kg/min管理的2uL/min率。这是一种高生理剂量。在可能的情况下，应努力限制输液量。胰岛素应适当稀释后，在血浆中存在的管理。 3％的生理盐水中大鼠血浆的解决方案是用来稀释在本实验室的胰岛素。胰岛素是HumulinR 100U/ml（礼莉莉），虽然可以采用其他快速长效胰岛素。确保稀释胰岛素混合之前，把输液注射器。 </li><li>准备50％葡萄糖。这可以注射器直接放入输液泵。标记"葡萄糖"泵"，以避免将来出现混乱。请记住，以确保足够数量的胰岛素和葡萄糖的研究。夹具一般〜2H。在本议定书中，3毫升注射器使用。 </li><li>到哈佛仪器11型加注射泵，将葡萄糖和胰岛素注射器。提前葡萄糖Y型连接用止血钳和夹钳线。事先胰岛素动物，钳止血线。钳线，让老鼠放宽为30分钟前开始实验。注:止血钳尖端警卫建议，以避免永久性线压着。 </li><li>准备离心的血浆采集和EDTA涂层管。在本研究中，将获得额外的血液样本，一旦动物被夹住。 </li></ol>第2部分:试验协议<ol><li>一个基线胰岛素样和血细胞比容样品应被收购。血细胞比容采样，确保整个实验过程中保持血容量。一般情况下，血细胞比容水平不应该下降超过10％的初始值。 </li><li>获取基线葡萄糖样本（单键超，LIFESCAN公司），并确定整个被夹住的血糖水平。在这里，我们在正常血糖钳，或5.0 - 5.5mm的（ 图2）。每个血液样本后，冲洗的10U/ml体积小动脉行肝素生理盐水，以防止凝血。注意:确保无凝块或气泡，在注入前行。这些可以给​​动物中风。 </li><li>启动胰岛素输注。在每个时间点的血糖监测血糖，以5 - 10分钟的时间间隔。调整葡萄糖输液速度，直到达到稳定状态。这个过程通常是一个试错的过程，可以采取30分钟&gt; 2H。须维持血糖水平的葡萄糖依赖于实验的协议，物种和条件（ 图2） 。 </li><li>稳态血糖水平的定义范围内的三个连续读数。在这里，〜1mm的内三读被认为是钳制（如4.8，5.2，5.6mM）。一旦达到稳定状态，记录为30分钟内必须保持血糖的葡萄糖输注率。在这段时间内，可以得到更多的血液样本。至少，第二血浆胰岛素样和血细胞比容样品应获得。 </li><li>钳完成后，可用于进一步的测试或安乐死，并作进一步的分析收集组织使用动物。根据动物，导管线可以保持5-7天的专利。 </li></ol> D:高胰岛素 - 正常血糖钳结果（胰岛素） 当正确执行，钳程序评估稳态大鼠胰岛素敏感性。在介绍钳从获得的数据，它是必不可少的文件血糖水平和葡萄糖输注率。稳定血糖水平超过最低的30分钟的时间（ 图3）当然是一个稳定的状态指示。葡萄糖被认为是只有全血葡萄糖〜1mm的范围内保持稳定。葡萄糖输注率表明外源性葡萄糖来维持血糖水平。在可能的情况下，这些数字应显示为一个时间过程，而不是单一，平均值（图4）。 建议报告的其他措施，血浆胰岛素和红细胞压积。空腹的决心和夹紧确认胰岛素水平，胰岛素已经成功地管理和将检测在治疗组之间的水平（图 5）任何差异。在基线和胰岛素钳夹结束时获得的血细胞比容措施建议（图 6 ）。这是为了确保血细胞比容水平不属于血液的流量和流速的实验，并在所附的改建期间超过5％，不会影响血糖处置。 <img src="/files/ftp_upload/2432/2432fig1.jpg" alt="图1" /> 图1实验装置。图1显示了在夹紧过程奔放，有意识地鼠。导管允许未经处理的动物的血液样本和输液。到左边的泵含有胰岛素和葡萄糖。 T"&gt; <img src="/files/ftp_upload/2432/2432fig2.jpg" alt="图2" /> 图2。有望在夹紧过程中的数据。为了获得钳在基准水平（血糖正常，5MM），外源性葡萄糖的水平（D50）操纵直到达到基线或"钳"。 <img src="/files/ftp_upload/2432/2432fig3.jpg" alt="图3" /> 图3:预期的钳制程序的血糖结果。当动物是"钳制"，血糖相对稳定，跨越了时间和实验组。 <img src="/files/ftp_upload/2432/2432fig4.jpg" alt="图1" /> 图4。代表的钳制程序的葡萄糖输注速率。要求，以保持血糖正常的外源性葡萄糖量是不同的。这是说明与控制（议员美联储）和高脂肪喂养动物（胰岛素抵抗）。高脂肪喂养的动物，需要较少的葡萄糖注入来维持血糖，主要是因为它是不敏感的胰岛素注入的。 <img src="/files/ftp_upload/2432/2432fig5.jpg" alt="图5" /> 图5代表对大鼠血浆胰岛素。血浆胰岛素在胰岛素钳夹应禁食，基线血浆胰岛素水平。这将确保胰岛素正常管理过程中的动物胰岛素钳夹。 <img src="/files/ftp_upload/2432/2432fig6.jpg" alt="图6" /> 图6。报告血细胞比容。以下实验的基线血细胞比容和红细胞压积，必须获得和报告。这将确保血细胞比容水平不下降超过5％的基准水平造成过多的动脉血取样。

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由卡尔加里动物保健大学和程序批准使用委员会和遵守加拿大的实验室动物科学实验的准则协会。作者有没有利益冲突或披露。

最初调查在人类胰岛素敏感性的发展，现在已经被钳制程序适应其他物种，包括实验大鼠和小鼠。调查胰岛素抵抗的动物模型提供了一个了解对胰岛素的敏感性和相关的病症的病理生理，以及确定的临床应用价值1,2的治疗性干预的大量援助。已受聘的几种方法，以评估在动物体内胰岛素的敏感性。这些技术包括糖耐量试验（GTTs）的变化和胰岛素糖耐量试验（ITTS）4-6以及?...

<table border="1"><tbody><tr valign="top"><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr valign="top"><td> Intramedic聚乙烯管（PE - 50） </td><td> Fisher Scientific则</td><td> 14 - 170 - 12B </td><td>内部直径0.58毫米（0.023"）的X外径0.965毫米（0.038"） </td></tr><tr valign="top"><td>道康宁的硅橡胶实验室套管</td><td> Fisher Scientific则</td><td> 11 - 189 - 15C </td><td>内部直径0.76毫米（0.030"）的X1.65毫米外径（0.065"） </td></tr><tr valign="top"><td>聚乙烯小号- 50 - HL医用导管</td><td>哈佛器械</td><td> PY2 72-1251 </td><td>内部直径为3.2mm（0.125"）的X4.7毫米外径（0.1875"） </td></tr><tr valign="top"><td>乐泰超级胶水</td><td>大和玩具</td><td> 32237 </td><td>凝胶控制</td></tr><tr valign="top"><td>无菌手术刀片</td><td> VWR </td><td> BD371610 </td><td></td></tr><tr valign="top"><td>弧形微解剖钳</td><td>乔治Tiemann＆CO。 </td><td> 160-20 </td><td> × 2 </td></tr><tr valign="top"><td>直微解剖钳</td><td>乔治Tiemann＆CO。 </td><td> 160-15 </td><td> × 2 </td></tr><tr valign="top"><td>弯止血钳</td><td>乔治Tiemann＆CO。 </td><td> 105-1135 </td><td> × 2 </td></tr><tr valign="top"><td>直止血钳</td><td>乔治Tiemann＆CO。 </td><td> 105-1130 </td><td> × 2 </td></tr><tr valign="top"><td>止血提示警卫</td><td>罗宾斯仪器公司</td><td> 15.09-2-004 </td><td></td></tr><tr valign="top"><td>直外科剪刀</td><td>乔治Tiemann＆CO。 </td><td> 105-402 </td><td></td></tr><tr valign="top"><td> VENOJECT多样品露儿适配器</td><td>泰尔茂医疗产品</td><td> 810127A </td><td> 21计，1英寸</td></tr><tr valign="top"><td>无菌导管导引</td><td>碧迪</td><td> 406999 </td><td></td></tr><tr valign="top"><td> 14号钝针</td><td>碧迪</td><td> 511310 </td><td> 14号，2英寸</td></tr><tr valign="top"><td>无菌手术缝合</td><td>强生公司医药产品</td><td> 1679H </td><td>丝绸，大小3-0 </td></tr><tr valign="top"><td>非无菌手术缝合线</td><td> Angiotech制药公司</td><td> SP116 </td><td>丝绸，大小4-0 </td></tr><tr valign="top"><td>棉签</td><td> VWR </td><td> 10806-005 </td><td></td></tr><tr valign="top"><td> 4ply纱布垫</td><td> VWR </td><td> CA43845 - 062 </td><td></td></tr><tr valign="top"><td>小动物无绳快船</td><td>哈佛器械</td><td> 729063 </td><td></td></tr><tr valign="top"><td>异氟醚</td><td>卤烃产品公司</td><td> IPN - 45 </td><td></td></tr><tr valign="top"><td>麻醉车</td><td>森医疗等行业，公司</td><td></td><td></td></tr><tr valign="top"><td> 70％的乙醇</td><td> Fisher Scientific则</td><td> HC - 1000 </td><td></td></tr><tr valign="top"><td>优碘消毒液</td><td>西药配送中心有限公司</td><td> 105267 </td><td></td></tr><tr valign="top"><td> 11型加注射泵</td><td>哈佛器械</td><td> 702208 </td><td></td></tr><tr valign="top"><td>不锈钢油管耦合</td><td>哈佛器械</td><td> 72-4434 </td><td> 23计，0.3英寸</td></tr><tr valign="top"><td>不锈钢管插头</td><td>哈佛器械</td><td> 72-4436 </td><td> 23计，0.5英寸</td></tr><tr valign="top"><td>不锈钢钝性针</td><td> Instech实验室，公司</td><td> LS22 </td><td> 22号</td></tr><tr valign="top"><td> 60度Y型连接器</td><td>小零件</td><td> STCY - 22 - 05 </td><td> 22号</td></tr><tr valign="top"><td> CritSpin微血细胞比容离心机</td><td>虹膜来样加工</td><td> CS12 </td><td></td></tr><tr valign="top"><td>迷你离心机</td><td> Fisher Scientific则</td><td> 05-090-100 </td><td></td></tr><tr valign="top"><td>微型离心管</td><td> VWR </td><td> 53550-778 </td><td></td></tr><tr valign="top"><td> 50毫升聚丙烯离心管</td><td> VWR</td><td> 89004-364 </td><td></td></tr><tr valign="top"><td> 1毫升塑料滑提示注射器</td><td>碧迪</td><td> 309602 </td><td></td></tr><tr valign="top"><td> 3毫升塑料Luerlok提示注射器</td><td>碧迪</td><td> 309585 </td><td></td></tr><tr valign="top"><td>肝素抗凝注射液</td><td>西药配送中心有限公司</td><td> 102824 </td><td>生产商:LEO制药公司浓。 1000国际单位</td></tr><tr valign="top"><td> EDTA溶液</td><td> Promega公司的公司</td><td> V4231 </td><td> 0.5米，pH值8.0 </td></tr><tr valign="top"><td>盐水</td><td>西药配送中心有限公司</td><td> ABB7983154 </td><td>制造商:Hospira公司0.9％的氯化钠</td></tr><tr valign="top"><td> 50％葡萄糖</td><td> Vetoquinol </td><td> 8DEX012D </td><td></td></tr><tr valign="top"><td>优泌林 - R </td><td>礼来公司</td><td> HI - 210 </td><td> 100U/ml </td></tr><tr valign="top"><td> 1毫升胰岛素注射器</td><td>碧迪</td><td> 309311 </td><td></td></tr><tr valign="top"><td> Fisherbrand *造血密封胶</td><td> Fisher Scientific则</td><td> 02-678 </td><td></td></tr><tr valign="top"><td> Fisherbrand * Microhematocrit毛细管</td><td> Fisher Scientific则</td><td> 22-362-574 </td><td></td></tr><tr valign="top"><td>单键超试纸</td><td> LIFESCAN，公司</td><td> AW 085 - 314H </td><td></td></tr><tr valign="top"><td>单键Ultra血糖仪</td><td> LIFESCAN，公司</td><td> AW 085 - 314B </td><td></td></tr><tr valign="top"><td>钠Pentobarbitol </td><td>塞瓦三特，石兽</td><td> 1715 138 </td><td>浓。 54.7mg/ml </td></tr><tr valign="top"><td>红实验室标签带</td><td> VWR </td><td> 89097-932 </td><td></td></tr><tr valign="top"><td>蓝实验室标签带</td><td> VWR </td><td> 89097-936 </td><td></td></tr><tr valign="top"><td>电子秤</td><td> Fisher Scientific则</td><td> 01-913-88 </td><td></td></tr><tr valign="top"><td>涡旋</td><td> VWR </td><td> 58815-234 </td><td></td></tr><tr valign="top"><td>定时器</td><td> VWR </td><td> 62344-641 </td><td></td></tr></tbody></table>

hyperinsulinemic-euglycemic clamp in the conscious rat

2型糖尿病（T2D）患病率迅速上升。其特点是无论是生产胰岛素不足或无法利用血糖水平升高的胰岛素产生，T2D结果。 "黄金标准"在评估胰岛素敏感性，是高胰岛素 - 正常血糖钳或胰岛素钳夹。在此过程中，胰岛素注入在一个恒定的，导致血糖下降率。为了保持血糖在一个恒定的水平，外源性葡萄糖（D50）注入静脉循环。注入的葡萄糖量保持动态平衡胰岛素敏感性的指标。在这里，我们展示在长期插管，奔放，有意识地鼠的基本钳程序。这种模式使血液收集，以最小的压力动物。麻醉诱导后，正中切口和左颈总动脉和右颈静脉插管。插入导管用肝素生理盐水冲洗，然后exteriorized和安全。动物被允许恢复实验前4-5天，每天监测体重增加。只有那些谁复胖到术前水平的动物用于实验。在一天的实验，老鼠被禁食含有胰岛素和D50泵相连。基线葡萄糖是从动脉行评估整个实验（血糖正常），并用一个基准。在此之后，是在一个恒定的速率进入静脉循环注入胰岛素。为了配合在血糖下降，D50的注入。如果D50的输液速度大于吸收率，血糖的上升会发生。同样的，如果利率是不够的，以配合整个身体对葡萄糖的摄取，下降会发生。葡萄糖的滴定继续进行，直到达到稳定血糖读数。血糖水平，在这个稳定的时期葡萄糖输注速率记录和报告。结果提供了一个全身胰岛素敏感性指数。该技术可以改进，以满足特定的实验要求。它进一步增强了使用的放射性示踪剂，可以决定组织特异性胰岛素刺激的葡萄糖摄取以及全身葡萄糖营业额。

Watch this Scientific Journal Video about Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat at JoVE.com

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

高胰岛素 - 正常血糖钳是胰岛素的作用进行评估的"金标准"。胰岛素是注入在一个恒定的刺激葡萄糖摄取率。注入外源性葡萄糖的量，以应对这一下降是胰岛素敏感性的指标。这里的程序是一种自觉的，奔放的大鼠上进行。

在自觉大鼠高胰岛素 - 正常血糖钳

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

hyperinsulinemic-euglycemic-clamp-in-the-conscious-rat

Research

JoVE Journal

Medicine

51.9K 次观看.  University of Calgary. 高胰岛素 - 正常血糖钳是胰岛素的作用进行评估的"金标准"。胰岛素是注入在一个恒定的刺激葡萄糖摄取率。注入外源性葡萄糖的量，以应对这一下降是胰岛素敏感性的指标。这里的程序是一种自觉的，奔放的大鼠上进行。

视频: Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

科研

医学

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

在缺血大鼠肺的血管生成

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜:对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

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. ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

该二甲基亚硝胺诱发肝纤维化模型大鼠

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

意识约束兔子中癫痫发作、心律失常和呼吸暂停的多系统监测

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...