Name: use of a central venous line for fluids, drugs and nutrient administration in a mouse model of critical illness
Uploaded: 2017-05-02T14:45:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness at JoVE.com

GVdB, via the University of Leuven (KU Leuven), receives long-term structural research support from the Methusalem Program funded by the Flemish Government (METH08/07) and holds a European Research Council Advanced Grant AdvG-2012-321670 from the Ideas Program of the European Union seventh framework program. SET received a Research Foundation-Flanders (FWO) Research Assistant Fellowship.

该方案经鲁汶伦理审查委员会的大学动物研究。 1.静脉管线的制备<ol><li>通过快速浸渍60厘米microrenathane（MRE）管在热（&gt; 220℃）芝麻油中间部分准备静脉导管的末端。接着伸展该管的中间部分通过轻轻移动管子的端部远离彼此，以产生一个窄的直径（外径（OD）&lt;0.5毫米）。 </li><li>使用手术刀片到管切成各30厘米（参见图1， 表1）两个部分。 </li><li>使用聚乙烯PE50连接器连接MRE油管聚乙烯管PE10和PE10此管路连接到旋转接头的底部。连接旋转到PE10管道的上部，该连接到与PE50连接器配管鲁尔存根针按照图1。 </li><li>应用强大的快速ACTI纳克粘合剂胶水于所有连接，并通过与空气冲洗测试泄漏的导管。气体消毒使用前导管。 </li></ol><img alt="图1" src="/files/ftp_upload/55553/55553fig1.jpg" /> 图1: 静脉线的建设。静脉线被拉伸MRE油管小的直径和通过聚乙烯管连接到一个啮齿动物回转装置制备。请参阅表1上不同部分的长度的指令。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55553/55553fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 传说 </td><td> 长度 </td><td> 卷 </td></tr><tr><td> 1）鲁尔存根针22G </td><td></td><td></td></tr><tr><td> 2）PE50 - 聚乙烯0.023&quot; ×0.038&quot; </td><td> 5厘米</td><td> 13μL </td></tr><tr><td> 3）PE10 - 聚乙烯0.011&quot; ×0.024" </td><td> 50厘米</td><td> 30微升</td></tr><tr><td> 4）啮齿类旋转20G的</td><td></td><td></td></tr><tr><td> 5）PE-10 </td><td> 15厘米</td><td> 9μL </td></tr><tr><td> 6）PE-50（连接器） </td><td> 5厘米</td><td> 13μL </td></tr><tr><td> 7）MRE025提示拉伸microrenathane 0.025&quot; ×0.012&quot; </td><td> 30厘米</td><td> 27μL </td></tr><tr><td>总和</td><td> 105厘米</td><td> 92μL </td></tr></tbody></table> 表1:静脉线的建设。此表提供了图1的图例。 2，麻醉和手术前处理<ol><li>使用24周龄的雄性C57BL / 6J小鼠（27 - 32克）。 注意:我们在24周大的小鼠（成熟的成年人），因为这年龄与相对应的重症监护患者的平均年龄为好。我们只使用雄性小鼠，避免雌激素的周期性影响。该技术还可以在较年轻的动物（在16周龄小鼠中测试）和女性使用时，如果优选的。 </li><li>在使用前消毒所有的手术器械。冲洗消毒自制静脉导管用生理盐水，以消除任何气泡和测试通畅和潜在的泄漏。 </li><li>切除部80厘米长的金属丝（0.8mm直径），通过折叠加倍，并进行循环。引线3个珍珠在一倍线到环路。通过经由3个珍珠引导导管的末端附接至导管的金属线。 注:金属附件线是必要的，以使导管被连接在旋转的旋转。这样，鼠标可以自由移动，而不会阻塞静脉管线。 </li><li>麻醉m个通过0.03毫升氯胺酮（100毫克/千克）和0.02毫升甲苯噻嗪（13毫克/千克）的混合物中的腹膜内（IP）注射乌斯。确保麻醉是通过检查没有反射的充足。轻轻拉动外鼠标舌钳通过舌头的吞咽，避免窒息。 注:如果在10分钟后，将小鼠仍试图撤出其四肢得到另外的氯胺酮作为0.01姆·博斯。 </li><li>剃手术区，包括腹部，颈部的腹侧（下巴，胸骨锁骨和之间三角形），和在所述头的基肩胛骨之间）。 </li><li>置于俯卧位鼠标上的预热的加热垫，消毒用70％乙醇对皮肤和应用少量眼科润滑剂的保护眼睛干燥。渗透到手术部位（腹部，颈部正面和背面）用0.2mL罗哌卡因（0.67毫克/千克）。 </li><li>而鼠标仍然处于俯卧位，作一小切口在背侧的基头与手术刀或剪刀。暴露颈后肌肉，并通过肌肉下导致了3.0的尼龙线与3.0的尼龙扎肌肉附接导线的环路。 </li><li>将在其右侧的鼠标。使腹侧颈部用手术刀或剪刀皮肤小切口垂直。下视觉引导，通过隧道向上穿过针先前制造的，穿入导管背面的切口此腹侧切口18号针头皮下注射至在腹侧exteriorize它。 </li><li>将在它的后面鼠标。通过鼠标和磁带下来的加热垫的顶部门牙后面的3.0尼龙线。在面罩用于输送氧气（2L /分钟）固定头。用胶带向下尾部和两个前肢固定在拉伸位置鼠标。 注:如果仅在氯胺酮 - 甲苯噻嗪麻醉不充分，吸入异氟醚（0.5 - 1.5％），可以在手术期间交付。 </li></ol><p类= "jove_title"&gt; 3。慢性留置导管置入<ol><li>鼠标放置在解剖显微镜下。在腹侧颈部以前所做的切口，轻轻挑逗脂肪组织和腺体远用钳子，直到颈静脉可视化。 </li><li>直截了当地解剖通过将静脉和结缔组织之间的钳子的前端，并与静脉并行地反复打开钳子释放从上方和周围的容器中的结缔组织和皮下组织的颈静脉中。这样，到静脉损害将被避免。 </li><li>通过放置钝钳静脉下分离颈静脉。订阅三块3.0丝线的静脉下，位置的一个片靠近所述颈静脉（颅结扎）和一个片靠近胸锁乳突肌（尾侧结扎）的分支。拧紧颅结扎和尾部结扎伸展静脉，防止出血过多而放置导管。 Ť即一个松散的结扎用中指螺纹，以确保以后的导管容易固定。 </li><li>采用微型剪刀，使沿头部和尾部连字符和大到足以通过导管之间的静脉切开。抓住用钳子导管，并插入导管11毫米进入静脉。松散地打结中间结扎固定导管并用无菌生理盐水冲洗轻轻确认正确位置。 </li><li>牢牢地拴住容器和导管周围的中部和尾部结扎固定导管。铁尾和中间结扎在一起，两端牢牢固定导管。确保结不闭塞静脉，通过冲洗导管每一个结发之后。最后，配合尾部和颅连字。关闭5.0丝线缝合切口。 </li></ol> 4.盲肠结扎和穿刺<ol><li>使通过与手术刀或剪刀腹部的下半部分的皮肤1cm的正中切口;注意不要渗入腹腔。 </li><li>确定腹部肌肉的白线，并做出间小切口，以获得进入腹腔。找到盲肠，并用钝解剖钳分离盲肠和exteriorize它。 </li><li>结扎盲肠在其与3.0丝缝线长度的50％。确保不要结扎回盲瓣，使肠道保持连续性。 </li><li>通过和 - 通过由单一的穿孔用18号针头盲肠结扎和盲肠的尖端之间穿刺中途。去除针之后，从孔挤出粪便少量以确保通畅。 </li><li>重新定位于腹腔的肠管，并关闭腹膜和皮肤用5.0丝线缝合。 注:平均而言，新学员要求10至15只动物，以能够顺利放置静脉导管，并在45分钟内完成CLP。经过培训期间，麻醉/手术RELA的10％的死亡率泰德可以预期。 </li></ol> 5.手术后治疗和液体复苏<ol><li>放置在仰卧位置的鼠标和导管固定到与条带附着导线。将鼠标移动到一个单独的笼子。使用具有调节夹具的支架来保持旋转装置25厘米鼠标在上面和牢固地用胶带将金属连接线连接到旋转接头的转动点的自由部分。 </li><li>附上包含平衡胶体和类晶体（1:4）的混合物用注射器向静脉线路开始液体复苏。 </li><li>放置在笼控制的温度（27°C）动物柜12小时光照和黑暗循环，并开始通过准确注射器驱动输液泵静脉内液体复苏（10毫升/千克/小时）。提供笼富集如排料和木块。 </li></ol> 6.重症监护<ol><li>在6小时后操作，皮下注射疼痛medication和抗生素（0.3毫升丁丙诺啡（0.15毫克/千克）和0.2毫升亚胺培南（16.67mg / kg）的）。注:按照标准实验室动物医学实践，按照该机构的兽医政策止痛药的第一个剂量在之前的初始手术切口，然后给出。皮下注射应仔细注射到皮下空间。 </li><li> 20个后 - 液体复苏的24小时，通过更换全肠外营养（6.67毫升/千克/小时）的类晶体/胶体。 注:全肠外营养给药（5.8千卡/ 24小时），占地约40％的小鼠，类似于早期的卡路里摄入量的病人在重症监护病房的每日热量需求。 </li><li>皮下给予止痛药和抗生素（0.6毫升丁丙诺啡（0.3毫克/千克）和0.2毫升亚胺培南（16.67毫克/千克）在危重病的整个期间，每12小时。 </li><li>检查动物至少每3小时白天。通过给予疼痛评估的痛苦基于经过验证的鼠标鬼脸规模11得分。加强与高疼痛评分监测动物。 </li></ol> 7.最终的实验注:批准并在模型和人类终点的方针，政策的严重级别建议应该从本地机构伦理审查委员会对动物研究的追捧。 注意:在非功能性静脉线路如封端的导管，该导管的离域的情况下，与注射泵的问题是，将动物从研究中排除和安乐死。 <ol><li>在实验期结束时，深深麻醉并用0.03毫升氯胺酮（100毫克/千克）和0.02毫升（13毫克/千克）赛拉嗪的混合物鼠标。通过心脏穿刺抽取血液安乐死鼠标。商店在-80℃抓拍感兴趣冰冻组织标本。 </li></ol>

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The authors have nothing to disclose.

我们开发重大疾病的更多的临床相关的小鼠模型中，通过结合盲肠结扎和穿刺方法诱发败血症与使用中心静脉导管为液体，药物和营养管理。该实验装置是可重复的，允许以研究重大疾病和结果的延长阶段稳定死亡率，特此模仿人类的临床情况1。 盲肠结扎和穿刺已经被广泛承认为再现败血症6,7的主要特征急性危重病的动物模型。然而，急危重症的这个模型不允许非解决多器官功能衰竭，肌肉萎缩，和内分泌及代谢的变化，这是典型的重大疾病的长期阶段的研究。另外，通过该模型的临床前研究结果经常无法translate将临床人类设定8，9，10。因此，我们开发了一个模型，其中放置中心静脉导管允许调查员来管理大量的液体复苏，药物和肠外营养。这些支持措施是对危重病人至关重要，为了生存重大疾病的急性期。尽管这个中心静脉导管的放置，动物仍然能够自由地与相对轻微不适移动。每日服用止痛药可以帮助减轻痛苦，疼痛11的鼠标鬼脸规模的评估。该模型还采用支持治疗的危重病人，如抗生素治疗和止痛药物等重要方面。 这种模式有一些局限性。首先，大型外科手术的要求很高，并且需要足够的培训。决不theless，有足够的训练，对手术的动物的平均10％的过程中或手术后立即死亡。此外，未充分放置导管，可导致复苏液或肠胃外营养的漏入动物的胸腔。平均而言，存活的动物的15％必须在研究过程中，由于这些导管相关的问题被排除在外。因此，当一个计算出所需的动物进行实验，人们必须考虑到由于手术和导管相关损失25％的盈余。其次，只有一个静脉接入点，抗生素和止痛药还是每天两次进行皮下注射。事实上，用抗生素和止痛药药品肠外营养的相容性不能得到保证，因此应避免共同输液。行之后是推注的冲洗也技术上不可能的，由于小鼠的有限血容量。三，麻醉和手术所需的牛逼Ó放置导管将引起由本身严重的应激反应。我们用健康的动物无需手术作为对照，并考虑导管相关手术的危重病的一部分，相当于人类什么外科ICU病人不得不忍受。四，而手术后止痛药和抗生素，并使用中心静脉导管的允许肠外营养的施用增加小鼠CLP模型的临床相关性，我们的模型仍然没有完全模仿人体临床情况。事实上，由于小动物的大小是有挑战性的为大家介绍几款支持先进技术，如肾脏替代治疗。然而，使用这种模式可以遗传的干扰，在揭示重大疾病的发病机制是至关重要的。 已经示出的CLP程序的死亡率和严重性可以被操纵，如果认为有必要，由被结扎的盲肠部，由大小和num穿刺的误码率，皮下液体复苏和每日施用抗生素6,12。我们选择在此我们以10mL /千克/小时的第20个小时提供了广泛的液体复苏，因为它已被以前表明，这提高死亡率13的协议。如果长期危重病的模型所需的，而不是致死的模型提供了修改应纳入的协议。该小鼠模型使用中级CLP，静脉内液体复苏和抗生素治疗，以创造一个更加临床相关的重大疾病的模型中，如通过其模拟人类脓毒症1的存活率其延长存活曲线所示。在ICU病人人口，总人口，老龄化14。为了模仿人类的ICU设置更加紧密，人们可以使用成熟的小鼠（6个月），在本研究中使用，或DER提升实验模型的临床意义。 总之，这种小鼠模型需要动物的大型外科手术和日常精心呵护，但它会导致重大疾病的模型，它允许研究者研究重大疾病的长期阶段的各个方面。

重大疾病是其中的一个或多个器官系统的功能受到阻碍该患者会死亡，除非强化药物载体施用的程度的疾病状态。而对于进入重症监护病房（ICU）可以改变，从外伤，手术复杂，烧伤，病情加重败血症的最初原因，所有的危重病人从细胞损伤，引起缺血，缺氧和过度炎症反应等遭受，从而导致器官衰竭。大多数患者生存的严重侮辱，但患者的一个重要部分不会马上恢复，并且需要长时间的重症监护室。他们在死亡的高风险，由于非解决多器官功能衰竭。此外，重大疾病的延长的相位被深刻肌肉无力和内分泌及代谢变化，这的发病机理目前不完全理解纯度标记。 几个R取代odent模型在重症监护研究环境中使用。两个最常用的模型是脂多糖（LPS）和盲肠结扎和穿刺（CLP）的外源施用。这两种型号的开发，以模仿败血症的急性期，定义为造成失调的宿主对感染的反应威胁生命的器官功能障碍，和的主要原因接纳全世界ICU 1，2中的一个。该LPS模型有几个缺点，因为它只有短暂影响细胞因子的释放和动物3的血流动力学状态。与人类不同，啮齿动物也对内毒素特别耐以及使用内毒素的"高剂量"是必要的，以产生低血压和死亡率，特此进一步提高该方法4,5的有效性的担忧。其他模型，盲肠结扎和穿刺模型（CLP）设有后跟一个针穿刺通过和 - 通过盲肠的一部分的一个结扎。此过程会导致多种微生物腹腔感染组织损伤，随后是易位细菌进入血液舱。这会引发全身炎症反应和脓毒症的发展。该CLP模型已被广泛承认为再现败血症的主要特征急危重症的动物模型:hyperinflammation，血管舒张，低血压和心输出量增加6,7。然而，这种模式不允许非解决多器官功能衰竭，肌肉萎缩，和内分泌及代谢的变化，这是典型的重大疾病的长期阶段的研究。此外，最近的小鼠模型对重大疾病的有效性受到质疑，因为从小鼠模型的研究结果不能总是被转换为人类设置8，9，10。一种可能的解释可能是提供给危重人类患者的支持治疗从提供给危重小鼠的护理有很大的不同。 因此，为了更接近于人类的设置，并允许重大疾病的长期阶段的调查，我们开发了模仿急性重症监护给人类，如丰富的静脉输液和抗生素治疗的小鼠模型，并允许为了生存重大疾病，如营养支持的延长期来管理支持治疗。为此，我们适应CLP-引起败血症的小鼠模型，是败血症的金标准，并放置在中央静脉线使体液的营养和药物管理。

<table><tbody><tr><td>Buprenorphine</td><td>Ecuphar</td><td></td><td>Vetergesic 0.3 mg/mL</td></tr><tr><td>C57BL/6&amp;nbsp;</td><td>Janvier labs</td><td>C57BL/6JRj</td><td></td></tr><tr><td>colloids</td><td>Fresenius Kabi</td><td></td><td>Volulyte 6%</td></tr><tr><td>crystalloids</td><td>Baxter</td><td></td><td>Plasmalyte a viaflo</td></tr><tr><td>Ethilon 3.0</td><td>Ethicon</td><td>F3211</td><td></td></tr><tr><td>Imipenem</td><td>MSD</td><td></td><td>Tienam 500&amp;nbsp;mg powder for injection fluid</td></tr><tr><td>Isoflurane</td><td>Eurovet</td><td></td><td>Iso-vet</td></tr><tr><td>Ketamine</td><td>Eurovet</td><td></td><td>Nimatek 100 mg/mL</td></tr><tr><td>LocTite Super glue3 all plastics&amp;nbsp;</td><td>Rectavit</td><td>119818</td><td></td></tr><tr><td>Mersilk 3.0</td><td>Ethicon</td><td>L192</td><td></td></tr><tr><td>Mersilk 5.0</td><td>Ethicon</td><td>F682</td><td></td></tr><tr><td>Microrenathane .025&quot; O.D. x .012&quot; I.D.</td><td>Bioseb</td><td>MRE-025</td><td></td></tr><tr><td>olimel N7E</td><td>Baxter</td><td></td><td></td></tr><tr><td>PE10 - Polyethylene .011&quot; x .024&quot;&amp;nbsp;</td><td>Instech Solomon</td><td>BTPE-10</td><td></td></tr><tr><td>PE-50 tubing .023&quot; x.038&quot;</td><td>Instech Solomon</td><td>BTPE-50</td><td></td></tr><tr><td>Rodent Swivel 20 G</td><td>Bioseb</td><td>RS-20G&amp;nbsp;</td><td></td></tr><tr><td>Ropivaca&amp;iuml;ne</td><td>Astrazenica</td><td></td><td>Naropin 2&amp;nbsp;mg/mL</td></tr><tr><td>Xylazine</td><td>VMD</td><td></td><td>Xylazine hydrochloride 2%</td></tr></tbody></table>

use of a central venous line for fluids, drugs and nutrient administration in a mouse model of critical illness

本协议描述长期重大疾病的中央插管小鼠模型。我们结合盲肠结扎和穿刺方法诱发败血症与使用中心静脉导管为液体，药物和营养管理，以模仿人类临床。危重病人需要为了生存密集的医疗支持。虽然大多数的患者会在几天之内恢复，有关的四分之一患者需要长期的精心护理，并在非解决多器官功能衰竭死亡的高风险。此外，重大疾病的延长的相位被深刻肌无力，和内分泌和代谢的变化，这的发病机理目前不完全理解纯度标记。在重症监护研究中最广泛使用的动物模型是盲肠结扎和穿刺模型诱发败血症。这是一个非常可重复的模型，类似于人类九月急性炎症和血液动力学变化SIS，其目的是研究重大疾病的急性期。然而，这种模式是由高杀伤力，这是从临床的人情况不同，并没有发展到研究重大疾病的长期阶段纯度标记。因此，我们通过将中心静脉导管在颈静脉使我们能够管理临床相关的支持性护理适应的技术，以更好地模仿重大疾病的人体临床情况。这种小鼠模型需要动物的大型外科手术和日常精心呵护，但它会导致重大疾病的急性和长期阶段的相关模型。

C57BL / 6小鼠，如上所述制成危重。我们进行了两个实验，以评估CLP后存活直到两个时间点:存活，直到第5天（N = 15）和存活直到第7天（N = 22）-CLP后。两个实验的存活曲线没有显著不同（比较，直到第5天），这表明实验装置的再现性。非存活，动物被发现死亡或安乐死由于人类到达终点。在用抗生素，通过在颈静脉的静脉导管液体复苏和全肠外营养组合盲肠50％结扎，如所描述的，导致了13％的死亡率1天后危重病，后3天24％的死亡率危重病，27 - 后危重病5天后31％的死亡率和7天后重大疾病的36％。健康成对饲养的小鼠，这是热量限制在危重的营养摄入患病小鼠没有表现出任何的死亡率。 <img alt="图2" src="/files/ftp_upload/55553/55553fig2.jpg" /> 图2: 存活曲线后重大疾病的5或7天。有组健康动物中没有死亡（A，B，虚线-健康的动物无需手术）。手术（A）五天后，死亡率为27％（实线）。 败血症（B）的诱导后七天，死亡率为36％。与泄漏或移出导管小鼠排除在实验（15％）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55553/55553fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness at JoVE.com

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

在重大疾病的小鼠模型中使用中心静脉导管的流体，药物和营养管理

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

use-central-venous-line-for-fluids-drugs-nutrient-administration

Nanyang Technological University

Research

JoVE Journal

Medicine

8.9K 次观看.  KU Leuven. This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. 

视频: Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

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

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

神经科学

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. ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

免疫与感染

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

持续静脉注射是精氨酸加压素受体拮抗剂考尼伐坦的选择治疗途径小鼠来研究中风诱发脑水肿

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

小鼠静脉内膜氧化术

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

中央静脉狭窄病的Murine模型,使用具有流出狭窄病的动脉静脉瘘管病

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

血管紧张素II诱导的腹主动脉瘤小鼠模型中中心静脉导管的药物治疗及3D超声监测

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

缺氧诱导小鼠肺动脉高压模型中的多次静脉推注给药和侵入性血流动力学评估

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...