作者要感谢俄亥俄州立大学实验动物资源中心（ULAR）的贡献。

健康的家养仔猪（ Sus scrofa）来自俄亥俄州立大学体育动物护理和使用委员会（IACUC）批准的农场。所有动物实验均按照俄亥俄州立大学IACUC政策执行，经议定书批准。 动物和动物处理<ol><li>在本实验中使用雄性仔猪消除性别的潜在混杂效应。注意:如果实验目标包括在最大脑生长期间评估实验对动物的影响，请勿使用长于14天的仔猪。 </li><li> 安排仔猪在实验前至少24小时到达生殖群，以适应环境。 注意:受过授权的兽医监督的经过培训的兽医技术人员提供常规动物护理。 <ol><li>将仔猪保存在各个温度控制的笼中，并给予营养商品小牛奶可随意购买。为动物提供毯子和玩具。连续监测动物围墙的温度。 </li></ol></li><li>对于这项初步可行性研究，为对照组使用18只小猪为异氟烷臂和22头仔猪。在可能的基础上进行基于研究设计的样本量计算。将可利用的仔猪随机分配给对照组或暴露组，以适当的曝光时间。从经验来看，即使有多名调查员，也希望能够每天不超过2次实验（总共2只动物）。 </li></ol>控制动物<ol><li>不要对对照动物进行实验干预。 </li><li> 通过面罩进行深度全身麻醉灌注和组织采集。具体来说，在24小时适应期后，用100％氧气中的5％异氟烷或8％七氟烷麻醉仔猪，通过面锥罩。不要使用地氟烷进行感应。 注意:诱导麻醉和冷PBS灌注的时间应尽可能短。经验丰富的技术人员可以在5分钟内完成此过程。 <ol><li>通过使用手术夹钳缺乏露水夹点反射来确认足够的麻醉深度。 </li><li>为了避免对大脑的缺氧/缺血性损伤，使用脉搏血氧计监测仔猪，以确保维持足够的氧合，通气和心输出量，直到冷磷酸盐缓冲盐水（PBS）灌注开始。 注意:为了提供额外的组织损伤保护，在诱导麻醉后将动物（包括头部）装入冰中。 </li></ol></li><li> 进行经心脏灌注。 注意:由于使用多聚甲醛，灌注程序应在通风橱或下降式桌子下进行。 <ol><li>做一个骷髅头我沿着胸骨的长度使用手术刀切割。切口的深度应足以暴露胸骨。 </li><li>仔细地用一把锋利的重型剪刀进行中线胸骨切开术，避免损伤心脏，肺或膈肌。如有必要，将手指放置在胸骨后部与胸腔内容物之间，以避免受伤。通过在隔膜中进行小（手指大小）的切口来操纵手指进入纵隔。 </li><li>进入胸腔后，用自锁式牵开器将肋骨保持打开状态。 </li><li>使用镊子和一把剪刀切开心包膜，暴露心跳。小心不要伤心。 </li><li>识别左心室，并仔细放置插管（如14 G血管导管）穿过心室的顶点。取下针头，将导管放在适当位置。 注意:注意不要刺穿心室后壁。来自导管的脉动血液回流指示它被正确放置。此时可以从动物中轻松取样血液。 </li><li>在确定右心房后，用剪刀在中庭大切口进行切开术，以便放血和排泄灌注液。 注意:通过吸入的异氟烷应持续进行，直到心脏死亡得到证实。通过直接观察到心输出量不足证实心脏死亡。 </li></ol></li><li> 使用由冷组成的灌注液灌注仔猪（4 ℃）含有浓度为5单位/ mL的肝素的磷酸盐缓冲盐水（PBS）。以每分钟300mL的速度冲洗5分钟，或直到溶液清洁。 <ol><li>注意灌注期间灌注插管不会脱落。使用市售的蠕动泵进行这种和所有其他灌注。 </li></ol></li><li> 进行半切除术移动脑的一个半球进行新鲜的组织分析。 注意:该协议允许检索新鲜脑组织的一个半球。另一个半球是固定的。如果不需要新鲜的纸巾，请跳至步骤2.6。 <ol><li>在此过程中，继续以每小时50毫升的速度循环冰冷的PBS，以确保大脑保持冷。 </li><li>沿着矢状缝合的长度在头皮中做一个纵向切口，直到使用手术刀的孔的大小。在此过程中，使用坚定的压力在头骨中创造一个分数。反映头皮暴露整个头颅。 </li><li>使用长颈鹿并从孔中开始，通过在颅骨和硬脑膜之间插入桡骨头，使用小心不要伤害下面的脑组织，从而将头骨移开。用骨头去除骨头，将其从脑实质中撬开。 </li><li>一旦颅骨被移除，切开并移除颅骨使用镊子和剪刀，再次小心不要伤害潜在的脑组织。 </li><li>在两个半球之间放置手术刀刀片，以小心地分开胼the体。 </li><li>使用扁平工具，如镊子的宽手柄末端，轻轻地收回额叶，逐渐切断颅神经，向前后方向工作。在半球的最后部，使用手术刀切割脊髓。移除整个半球。 注意:未固定的脑组织是脆弱的。取出半球时要小心，以防止剩下的半球血液供应中断。 </li><li>切除已移除的半球。如果指示，立即在液氮浴中冷却至-160℃的2-甲基丁烷中快速冷冻，以避免组织破裂，并立即储存于-80℃以备后续分析。 注意:我们建议使用矩阵以2毫米为单位进行冠状动脉切除冠状动脉，但需要特定的定位切片将取决于具体的实验目标。 </li></ol></li><li>将灌注液更换为4％多聚甲醛（PFA）。继续P300灌注，每分钟300毫升至少5分钟。 警告！ PFA有毒，避免与皮肤，眼睛或粘膜接触。不要吸入PFA烟雾。 </li><li> 期望由于在肌肉中形成醛交联而使仔猪的身体变硬。在PFA灌注完成后，以与步骤2.5.5中所述相同的方式去除剩余的半球。 注意:正确灌注的大脑会变苍白，完全放血。 <ol><li>将剩余的半球置于4℃的新鲜4％PFA的小容器中。保持半球在PFA 24-48小时完成固定过程。 </li><li> 24-48小时后，将固定的脑移动到含有0.1％叠氮化钠的PBS溶液中，因为防止过度固定是必要的。过度固定可能导致mas国王的表位或强非特异性背景染色。加入叠氮化钠可防止细菌生长。 注意:组织可以在4°C下储存长达一个月。 </li></ol></li></ol> 3.异氟烷（实验）动物注意:可以使用任何麻醉或干预措施，但我们不建议使用地氟烷进行吸入诱导。 <ol><li> 诱导和维持麻醉: <ol><li>使用装有儿科呼吸机和监测装置的临床麻醉工作站进行麻醉。 </li><li>在24小时适应期后，通过面锥罩将100％O 2中的8％七氟醚麻醉。 </li><li>在诱导期和研究过程中的任何时候，持续监测脉搏血氧饱和度，无创血压，心电图和温度。 </li><li>诱导后滴定七氟醚或异氟醚以达到足够的麻醉深度同时确保持续的自发呼吸（通常浓度为3-4％）的浓度。 </li><li>将24 G外周静脉导管置于边缘耳静脉（ 图1 ）。 </li><li>将仔猪置于背侧卧位，用于气管插管（ 图2 ，图A）。使用Miller＃1或＃1.5刀片，以便于气管下咽管插管。注意:在喉镜检查期间需要有经验的操作员和助手。 <ol><li>助手用干燥的纱布替代动物的舌头，以方便喉部的暴露和声带的可视化（ 图 2B ）。 注意:会厌仔猪的形态学类似于人类（ 图 2C ）。小猪唱歌公司ds可能难以可视化，因为它们在喉入口内几毫米深（ 图 2D ）。 </li><li>放置会厌:将喉镜尖端放在会厌下面，向上提起刀片以暴露喉。 </li><li>在将管置入气管之前，用0.5mL的2％利多卡因喷雾声带，以防止在气管插管通过期间的喉痉挛，因为仔猪特别容易发生喉痉挛。 </li></ol></li><li>放置并固定3.0毫米袖口气管插管。 <ol><li>使用听诊器和EtCO 2监测，使用胸部听诊确保双侧呼吸音和持续的潮气二氧化碳。 </li><li>将仔猪的肺充气至20 cm H 2 O的连续气道压力。然后，将气管内管的气囊充气至所需的最小压力，以防止压力为20 cm H的空气泄漏<sub&gt; 2 O，听取气管，以确定存在的空气泄漏。 注意:这对于在间歇性正压通气期间防止粘膜缺血是重要的。 </li><li>在麻醉过程中维持正常和正常的感觉。 </li></ol></li><li>在50％氧气/ 50％空气中开始给予2％异氟烷。滴定氧气以保持PaO 2为90-100mmHg。持续3小时（或期望的实验持续时间）。 </li><li>在眼睛上涂上眼科软膏，以防止麻醉期间的干燥。 </li></ol></li><li> 开始2％异氟烷后开始股动脉插管。 <ol><li>通过外周静脉注射管理广谱抗生素预切口（头孢唑啉，25 mg / kg） ，以防止手术部位感染。 </li><li>使用有色氯己定灭菌两只腹股沟，以确保适当的无菌田，并放置适当的无菌悬垂（ 图3 </strong&gt; B ）。 至少，参加生存手术的工作人员应佩戴手术帽，面罩，无菌手套和眼睛保护。 </li><li>使用指数和中指在腹股沟折痕处掌握股动脉。 </li><li>使用手术刀做一个浅表，1.5厘米的头骨切口（ 图3 C ）。 </li><li>使用钝器仪器（如手术止血钳或钝头剪刀）解剖粗糙肌肉的两个头部（ 图3 D ）。 注意:股骨神经血管束通常发现在这两只肌肉之间，并且仅深入这两只肌肉。 （ 图4A ）。 </li><li>使用血管环或丝带，隔离近端和远端的动脉。使用回路将动脉向上拉到皮肤水平（ 图4 B ）。 </li><li>而将牵引力放置在近端绑带上，足以中断血流，用一对十足剪刀做小动脉切开术。 <ol><li>注意不要横断动脉。小动脉切开术就足够了。或者，使用针和线方式进入动脉（ 图 4C）。 </li><li>近端扎带上的温和牵引力可以防止在手术过程中的任何一点出现过多的失血。如果使用针和线接近，请将导丝（随套件提供或单独获得）穿过针头并进入动脉至5厘米。 </li><li>注意不要进一步推进电线，因为它可能导致心室异位。如果发生这种情况，立即将电线拔出1-2厘米。 </li></ol></li><li>从容器中取出针头，注意将导丝线放在容器中。轻轻地将导管穿过导线并进入容器（ 图4） <strong&gt; D）。 <ol><li>使用3法国8厘米的导管进行股动脉插管术。当导管的尖端首先进入血管的浅表壁时，预期温和的阻力。 </li></ol></li><li>如果使用动脉切开术，将导管或聚乙烯管直接推入血管。应立即观察血液回流。 </li><li>立即将导管连接到压力传感器上。用生理盐水冲洗导管以维持导管通畅。 <ol><li>放置缝合线将导管固定到位。用无菌纱布覆盖切口以防止污染。利用经皮途径进行股动脉插管术。 注意:确保超声波由经验丰富的技术人员执行。 </li></ol></li></ol></li><li> 手术条件和监测: <ol><li>用强制空气加温装置对仔猪进行积极的保暖，并持续监测直肠温度（ 图3A ）。 </li><li>以维持率（猪仔重量，千克，mL / hr）维持率，将含葡萄糖的等渗液（林格氏乳酸盐或生理盐水中的5％葡萄糖）浸泡。 </li><li>监测扰动的生命体征（低血压，心律失常，低温/高热，缺氧） 注意:异常情况下，正常生命体征范围和相应建议的管理情况如表1所示 。 </li><li>使用市售的血液分析系统，在实验期间至少每小时测量动脉血气（动脉pH，pCO 2 ，pO 2 ），电解质（碳酸氢盐，碱过量/缺乏，钠，钾，离子钙），血红蛋白和葡萄糖期。从股动脉导管抽取动脉血样。 注意:正常酸碱和电解质值以及如果出现异常，则进行校正的建议重复总结在表1中 。 </li></ol></li><li> 3小时异氟烷暴露后，取出股动脉导管。 <ol><li>仔细绑扎近端血管丝以永久性闭塞股动脉以防止出血。或者，使用血管夹。 </li><li>在关闭切口前确保完全止血。用10-20毫升无菌盐水溶液冲洗切口，以防止感染。 </li></ol></li><li> 在实验结束时，使用3-0不可吸收的缝合材料，用简单的中断缝线闭合皮肤切口。 <ol><li>使用0.5-1 mL / kg 0.25％布比卡因和1:200,000肾上腺素浸润伤口，用于疼痛控制。用无菌的手术皮肤粘合剂涂上切口。 注意:不需要敷料。 </li></ol></li><li>停止麻醉，让仔猪醒来。 </li><li>在awa的迹象上移除气管内管开心（眼睛开放，试图站立，踢，打开和关闭嘴巴），具有稳定的血液动力学，足够的氧合和足够通气的迹象。 </li><li>拔管后，通过面锥补充氧气，直到充足的氧合和通气得到保证。 </li><li>皮下注射丁丙诺啡0.05 mg / kg，进行更多的疼痛控制。或者，可以使用透皮芬太尼。 </li><li> 在适当的时候，将仔猪送回家中。在重新获得足够的意识以维持胸骨躺卧之前，不要离开小猪无人看管。不要将动物带回其他动物的笼子，直到它完全从麻醉中恢复。 <ol><li>用温暖的灯光积极地加热私人家庭笼子。 </li><li>训练有素的兽医或研究人员在麻醉后密切监测动物。提供仔猪奶替代品。 </li></ol></li><li> 让仔猪恢复48-72小时，取决于实验目标。 <ol><li>根据需要每3小时皮下注射丁丙诺啡，以确保疼痛控制，经验丰富的动物护理人员可以控制动物的手术后不适感染皮下注射仔猪。 </li><li>在手术后的头6h，每4小时监测一次仔猪。以相同的方式进行动物牺牲和组织采购以控制上述动物。 </li></ol></li></ol>

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作者宣称他们没有竞争的利益。

关键协议步骤/故障排除随着实验开始，非侵入性生命体征的监测应从诱导开始。可以容易地获得和监测血压，心率，氧饱和度和直肠温度。仔猪应该在一个暖气装置下保持足够的核心体温，因为这些动物可以在全身麻醉下迅速变得低温。外周静脉导管的及时放置允许在诱导期间出现紧急情况。重要的是在整个异氟烷过程和牺牲程序中连续监测小猪，非侵入性或侵入性。在整个方案中，仔猪可以在多个步骤中，特别是在气道管理和插管过程中，能够非常快速地体验动脉氧饱和度。我们使用8％的七氟醚诱导麻醉，以复制人类儿科实践和刺激d感应。但是，5％异氟烷已经成功使用并且是合适的。鉴于解剖学上的差异和咽喉痉挛的倾向，仔猪难以插管。如果在诱导和/或气道管理期间小猪开始脱饱和，则应立即通过面锥管理100％的氧气和七氟醚，以便重新建立安全的氧饱和度水平和足够的麻醉深度。记住，当麻醉平面必须足够深以允许插管时，过度麻醉可能导致呼吸暂停。需要对动物的通气和氧合持续警惕，并相应地滴入吸入麻醉剂。一旦氧合恢复并且已经达到充分的麻醉，则可以重新插管插管。可以尝试通过面锥进行正压通气，但通常不成功。如果发生喉痉挛，将利多卡因溶液直接施用于voc指示允许气管插管。 紧急药物应始终可用，并且应在协议的关键部分根据需要进行治疗以纠正生理紊乱。尽管仔细讨论了仔猪的麻醉和紧急药物使用不在本手稿范围之内，但Swindle的"实验室中的猪:手术，麻醉，成像和实验技术"是一个很好的资源。 27 类似地，在中线胸骨切开术期间打开胸腔后，仔猪可以在牺牲期间开始快速去饱和。操作者应尽快工作，安全地暴露心脏并插入血管导管以开始冷PBS。需要彻底的冷PBS灌注（如果指示使用PFA快速固定），以防止对脑的缺血性损伤。 一旦仔猪插管，呼吸开始进行压缩率和尾气二氧化碳跟踪（ 表1 ）。通过滴定呼吸机支持同时保持足够的麻醉来稳定仔猪的潮气充氧和通气。我们使用机械通气来尽可能地模拟人类使用的物质。应避免高氧，以尽量减少氧化应激的机会。 异氟烷仔猪经历股动脉插管有两个原因:持续监测动脉血压;并在整个程序中采集动脉血来评估酸碱状态，血气和电解质。股动脉的插管是有挑战性的。请参阅影片的详细资料。对于生存实验，该程序应在无菌条件下的无菌操作环境中进行。在插入股动脉后，开始每小时监测动脉血气和血清电解质，根据需要进行校正维持体内平衡（ 表1 ）。小猪应接受含葡萄糖的连续等渗液以维持足够的血糖。在整个实验过程中，应持续监测动物的体温，并根据需要提供强制空气加温。避免体温过低和高热也同样重要。 虽然这个协议提供了一个"新鲜"脑半球和固定神经组织的一个半球，但这可以很容易地适应于替代的研究设计。也可以从仔猪收集额外的样品。在仔猪麻醉后可以获得CSF，有或没有荧光透视指导。也可以在方案的各个阶段从包括股动脉导管在内的小猪收集血液，以及在灌注之前立即通过血管导管从左心室收集血液。恢复期也可以延长或减少<html>分别用于检查慢性或急性反应。 技术的局限性该协议和模型在技术上是具有挑战性的。需要熟练的调查员和完全提供的操作套件，特别是对于生存实验。调查员（和助理，对于协议的某些部分）必须对本协议的手术和麻醉成分都感到舒适，这可能需要培训和经验来掌握。其他限制包括仔猪相对于啮齿动物模型的费用，尽管仔猪模型比非人类灵长类动物成本低得多。虽然仔猪的成本将根据获得动物的地区和农场而有所不同，但可以预期每只动物的成本低于500美元，而非人类灵长类动物的数量可以是每只动物数千美元。根据我们的经验，每只动物的平均成本通常约为200美元。 最后，由于仔猪模型的目的是模仿发育中的人类大脑，所以只能使用新生儿仔猪，中枢神经系统在快速生长期间是最脆弱的，而在仔猪期间，这一时期从出生后六周，出生后五周8 ，使用距离分娩日期更远的较长的仔猪，有降低仔猪模型临床意义的风险，而对于仔猪大脑发育的"等效性"则存在重大争议人类和猪的早期出生后大脑发育相比，人类新生儿的发育有惊人的相似之处。人类和猪的大脑分别是成人体重的27％和25％。14基于约翰逊的工作和同事，我们可以推断出一个仔猪周大致相当于一个人月。9这些结果基于wh<html>油脑体积数据，已经通过Workman及其同事的工作验证。 28我们选择了7-14日龄的仔猪，以估计1-2个月的人口。然而，如果实验目标是模拟仔猪脑生长发育的顶峰，可能审慎使用年轻动物（1-5天龄）。这是可行的，因为仔猪可以在出生时断奶。我们使用仔猪模型将适应新的数据可用于人与猪出生后大脑发育的相似之处。 关于替代/现有方法的技术意义仔猪与人类新生儿具有惊人的相似之处，包括脑部发育和病理生理反应中的关键平行。因此，它是临床相关的哺乳动物模型，概念验证研究表明，仔猪是麻醉神经毒性研究的合适模型参考文献"29,30 ，也可以轻松适应其他类型的发展神经科学研究，该模型旨在通过科学权威调查AIDN的程度和机制，最大限度地减少混合因素（如缺氧或高碳酸血症）的担忧导致麻醉引起的神经损伤，为了达到这个目的，仔猪用相同的手术和麻醉条件和儿科病人的监测进行治疗。 掌握技术后的未来方向和应用向前走，仔猪也非常适合神经认知测试17 。这个属性将允许对未来实验中麻醉剂暴露后的神经认知结果进行复杂，全面的评估。还应该强调的是，在临床环境中，儿童最常接受麻醉生理压力程序（手术）。麻醉和手术后炎症之间的相互作用以及所产生的神经元损伤和/或毒性（如啮齿动物和灵长类动物中所见）值得进一步探索和重要考虑。新生仔猪提供了一个独特的临床相关的基线模型，用于麻醉剂对发育中大脑的影响，而没有手术的混杂影响（模仿儿童常见的临床情况）。现在可以使用该模型可靠地测试不同类型手术或其他混杂因素（缺血，脑损伤，遗传易感性等 ）的影响。 在实验室，我们计划使用多种电生理和电化学方法进一步研究完整神经回路中麻醉和AIDN的机制。这些技术包括体内测量神经递质活性，全细胞膜片钳记录，神经成像和脑切片中的神经生理学研究。对于未成熟脑中的神经科学，与非人类灵长类动物存在很少缺点的小鼠模型相比，仔猪与人相关。随着进一步发展，仔猪可能是人类发展神经科学研究的理想模式。

每年，美国数百万儿童接受全身麻醉，其中许多儿童在4岁以下。麻醉诱发的发育神经毒性（AIDN）已经成为儿科麻醉研究的重点，因为了解麻醉对未成熟大脑的影响已成为当务之急。以前的研究表明，常用的麻醉药，如异氟烷，会引起年轻动物大脑神经细胞凋亡增加。对儿童的研究产生了不明确的结果2 。了解AIDN的发病机制，确定其预防或改善的潜在治疗靶点，并描述可用的最安全的麻醉方案已成为儿科麻醉社区的紧迫目标。本研究的主要目的是开发一种用于量化麻醉剂对发育中大脑的影响的最佳动物模型和方法，并仔细地刺激设计调查目前广泛使用的麻醉剂的安全性。 在最近对AIDN临床前文献的系统综述中，作者指出了超过900项研究中的重大方法异质性3 。许多人认为这是一个呼吁临床相关，精心设计的临床前模型，尽管已经对这个问题进行了多年的研究，但尚不存在。绝大多数啮齿动物模型使用不允许严格生理监测，血液取样或机械通气的方法。由于大脑对生理紊乱非常敏感，很难依赖这些模型的结果。开发该模型的主要目的是设计它，以便在需要时监测和纠正所有生理变量，如血气参数，体温，呼吸参数等 。 4 。翻译小猪模型提供了在这些评论和社论中寻求的临床相关性水平，因为它旨在解决这一需要，可以为未来的临床研究提供相关的临床前数据。 异氟烷是一种GABA A型（GABA A ）受体激动剂和弱NMDA受体拮抗剂，是全球临床实践中常用的吸入麻醉剂。只要不引起低血压或缺氧，异位异构体的麻醉剂就被认为是安全的;但是，可能会发生更微妙的影响。当大脑暴露于全身麻醉时，GABA的平衡aGonism和NMDA拮抗作用被破坏，导致细胞结构，连通性和功能的改变。此外，虽然GABA通常是抑制性神经递质，但已知在未成熟的脑中是兴奋的5 。正确地，当GABA从兴奋性转变为抑制发生时，尚不清楚，并且很可能是物种依赖性的。 当在所谓的"大脑生长突发"期间发生大脑兴奋性和抑制性输入之间的不平衡时，所产生的关键分子途径的兴奋性毒性失调可能导致异常的神经发育，例如凋亡性神经变性。除增加细胞凋亡外，氧化应激和炎症也可能被诱导，而神经元细胞增殖，神经元迁移和轴突核移动被抑制或失调6 。最终的结果是可能持续到adul的神经认知障碍你2 为了直接测量异氟烷对年轻哺乳动物的神经毒性作用，使用新生仔猪。与其他哺乳动物相比，小猪与人类有更多的CNS相似性，因此，他们的神经发育和神经解剖学相似性使它们成为临床相关的AIDN哺乳动物模型的理想动物。人类和仔猪都拥有脑卒中的脑，在脑回旋，灰质和白质的特征和分布中分享相似之处。小猪海马，基底神经节和脑干也与人类的形态相似7 。发育上，仔猪是经历围产期脑部生长和髓鞘形成的少数非人类哺乳动物之一8 。在子宫内，人和仔猪的大脑在妊娠晚期都经历显着的生长。在相关性方面，出生时，人和小猪的大脑分别是成人脑的27％和25％。磁共振成像显示，一周龄的仔猪大脑相当于一个月大的人脑，在神经元成熟和树突状态下9 。此外，仔猪和人脑在神经发育模式方面有许多相似之处。例如，卷线蛋白10的表达和mRNA序列，5-HT神经元11和神经管闭合12的形貌分布全部与人类相似。此外，仔猪和人类的基因组之间存在广泛的同源性。 动物模型的相关性必须在人类病理学的背景下被理解，特别是关于人类婴幼儿的脑成熟和病理生物学。大多数现有麻醉毒性研究使用啮齿动物模型，少数使用非人类灵长类动物模型。然而，啮齿动物和灵长类动物可能不是调查艾滋病的理想动物。 虽然广泛使用，但在发展过程中，啮齿动物的大脑与人类大不相同。最值得注意的是，啮齿动物具有脑脊液（或平滑）的脑。啮齿动物的大脑缺乏更神经复杂的生物体的特征的回旋和ul。。啮齿动物的大脑也经历了与人类和仔猪不同的出生后大脑生长突变14 。已经观察到，不同大脑区域对吸入麻醉剂的脆弱性有差异。因此，重要的是研究AIDN的动物模型具有神经发育和神经解剖学上类似于人类的脑，以便最好地模拟在儿科患者中可能看到的麻醉诱发的脑部改变。如前所述，仔猪拥有一只大脑，更适合这个角色。此外，啮齿动物神经认知测试的常见形式，如Morris水迷宫中评估的空间学习和记忆，并不直接相关或与幼儿的神经认知评估相媲美。使用仔猪进行发育神经科学的一个优点是，即使在小时候，它们也非常适合进行神经认知测试。被认为对其他哺乳动物物种有用的许多神经认知测试已经在猪中得到成功应用和验证。虽然仍然是一个不断发展的领域，仔猪的神经认知评估包括更复杂的测试，更好地模仿人类的缺陷，如斜梁运动测试17,18和镜面空间意识测试19 。作为小猪创伤性脑损伤研究的一部分，倾斜梁的电机测试在评估中显示出高可靠性的运动功能。镜像测试显示了周围环境的记忆，加上识别和利用反映的图像来寻找食物奖励。 另一方面，非人灵长类动物可能是儿科麻醉研究的更合适的模型，但是有一些禁忌因素，包括成本和使用难度。此外，它们对早期养殖条件非常敏感，特别是压力和母亲分离20 。对于AIDN研究重要的因素，如变构调节剂，受体 - 配体亲和力，翻译后修饰，受体亚单位组成和可变剪接变体，在灵长类动物的情况下是未知的。这是因为与这些概念相关的基因尚未克隆。相比之下，它们已被克隆在猪中。因此，在非人灵长类动物21,22 中仅进行有限的工作</suP&gt;。 仔猪模型利用了啮齿动物和非人类灵长类动物模型的优点:相对于非人类灵长类动物研究，它具有成本效益，易于使用，并且在神经解剖和神经生理学上类似于儿科人类大脑。近年来，使用仔猪进行神经科学研究已有所增加，其中包括一些研究小儿神经炎症状况的研究。呼吸道病毒感染对海马和空间学习的影响23 ，减轻脑卒中后脑细胞死亡24 ，创伤性脑损伤后的神经发生25 ，癫痫发作期间的酶活性26是使用新生仔猪的一些研究。这种大量和不断增长的文献体现了临床相关性和高度可重复性的仔猪研究麻醉方式的适用性和可持续性的强度sia诱导的神经毒性。

<table border="0" cellpadding="0" cellspacing="0" width="1164">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liqui-Wean</td>
			<td style="width:204px;">Milk Specialities</td>
			<td style="width:141px;">454836</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Piglet Anesthesia Face-Cone Mask</td>
			<td style="width:204px;">VetEquip</td>
			<td>921428</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Masterflex L/S Peristaltic Pump</td>
			<td style="width:204px;">Cole-Parmer</td>
			<td>EW-77916-20</td>
			<td style="width:516px;">Alternative peristaltic pumps can be used, as long as a constant and sufficient perfusion rate can be achieved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Masterflex L/S Pump Tubing, 25ft</td>
			<td style="width:204px;">Cole-Parmer</td>
			<td>EW-96410-24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">14-gauge angiocatheter</td>
			<td style="width:204px;">Becton-Dickson</td>
			<td>381164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">10X PBS</td>
			<td style="width:204px;">Thermo-Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Paraformaldehyde powder</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>P6148-5KG</td>
			<td style="width:516px;">Our lab makes this reagent from the powder as it is much more cost-effective. Prepared paraformaldehyde can also be purchased.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2-methylbutane</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>M32631-4L</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Needle holder</td>
			<td style="width:204px;">Teleflex</td>
			<td>152720</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Right angle clamp</td>
			<td style="width:204px;">Teleflex</td>
			<td>496217</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Rongeurs</td>
			<td style="width:204px;">Teleflex</td>
			<td>028120</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tenotomy scissors</td>
			<td style="width:204px;">Teleflex</td>
			<td>423480</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stitch scissors</td>
			<td style="width:204px;">Teleflex</td>
			<td>423440</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">McPherson Tying Forceps</td>
			<td style="width:204px;">Teleflex</td>
			<td>425200</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Adson Tissue Forceps</td>
			<td style="width:204px;">Teleflex</td>
			<td>181223</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">3-0 nylon suture</td>
			<td style="width:204px;">Medline</td>
			<td>ETH627H</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:303px;">Integra SL Anesthesia Workstation</td>
			<td style="width:204px;">DRE Veterinary</td>
			<td style="width:141px;">2350</td>
			<td style="width:516px;">This anesthesia workstation is chosen to best mimic the clinical monitoring experienced by pediatric patients in the operating room. Any anesthesia machine can be used as long as it allows for sufficient physiologic monitoring and intervention.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:303px;">Laryngoscope handle</td>
			<td style="width:204px;">Teleflex</td>
			<td align="right">8710000</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:303px;">Miller 1 Laryngoscope blade</td>
			<td style="width:204px;">Teleflex</td>
			<td align="right">2216100</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bair Hugger</td>
			<td style="width:204px;">3M</td>
			<td style="width:141px;">750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bair Hugger Torso Blanket</td>
			<td style="width:204px;">3M</td>
			<td style="width:141px;">540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">iStat Handheld</td>
			<td style="width:204px;">Abbott Point of Care</td>
			<td style="width:141px;">300</td>
			<td>Alternative point of care arterial blood gas analysis devices may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">iStat Cartridges</td>
			<td style="width:204px;">Abbott Point of Care</td>
			<td style="width:141px;">CG8+</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Dermabond Advanced Topic Skin Adhesive</td>
			<td style="width:204px;">Ethicon</td>
			<td>DNX6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LMA Laryngotracheal Atomization Device</td>
			<td style="width:204px;">Teleflex</td>
			<td>MAD720</td>
			<td>A cotton-tipped applicator soaked in local anesthetic can also be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Sheridan CF 3.0 Cuffed Endotracheal Tube</td>
			<td style="width:204px;">Teleflex</td>
			<td>5-10106</td>
			<td style="width:516px;">This model ETT was selected because it has a Murphy&#39;s eye, which is important to prevent ETT occlusion during the experiment</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:303px;">Pediatric Intubation Stylet</td>
			<td style="width:204px;">Smiths Medical</td>
			<td>100/120/100</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">24-gauge angiocatheter</td>
			<td style="width:204px;">Becton-Dickson</td>
			<td align="right" style="width:141px;">381112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">#10 Disposable Scalpel</td>
			<td style="width:204px;">Ted Pella, Inc</td>
			<td style="width:141px;">549-9-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Arterial Pressure Monitoring Kit 
			(3 French, 8 cm catheter)</td>
			<td style="width:204px;">Cook Medical</td>
			<td>C-PMSY-300-FA</td>
			<td>Simple polyethylene tubing with a luer-lock adapter can also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Intramedic PE90 Polyethylene tubing</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td style="width:141px;">14-170-12D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Monoject Blunt Cannula</td>
			<td style="width:204px;">VWR International</td>
			<td style="width:141px;">15141-144</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a piglet model for the study of anesthetic-induced developmental neurotoxicity (aidn): a translational neuroscience approach

在许多情况下，特别是儿童需要手术时，麻醉是不可避免的。最近对动物的研究引起了人们的担忧，即麻醉暴露可能导致神经元凋亡，称为麻醉诱导的发育神经毒性（AIDN）。此外，一些儿童临床研究表明，麻醉暴露可能会导致生命后期的神经发育缺陷。然而，临床前研究的理想动物模型尚未形成。新生小猪代表了临床前研究的一个有价值的模型，因为它们与人类具有许多发展相似之处。 仔猪的解剖和生理学允许在生存和非生存过程中实施严格的人围手术期条件。股动脉插管术允许密切监测，从而能够及时纠正仔猪生命体征和化学物质的任何偏差。一世此外，仔猪和人类新生儿之间存在多重发育相似之处。使用仔猪进行实验所需的技术将需要掌握经验。儿科麻醉师是调查小组的关键成员。我们在一般意义上描述了适合使用仔猪模型进行神经发育研究。

<html>研究了40头仔猪（18个异氟烷，22个对照）。所有动物的研究程序都能很好地耐受。所有仔猪都是男性。各组之间在年龄或体重方面差异无统计学意义（ 表2， 图5 ）。在异氟烷组实验过程中的平均实验室值见表3 。这些值表明，实验方案具有内部一致性和重复性，因为我们有多名技术人员在两年内进行手术。没有这些数字是我们在手术过程中进行的许多调整和修正，以维持生理止血。 CO 2保留，低核心体温和低血糖是我们通过综合监测和调整所必需的许多混淆因素中的一些。 t"fo:keep-together.within-page ="1"&gt; <img alt="图1" class="xfigimg" src="/files/ftp_upload/55193/55193fig1.jpg" /> 图1:。外周静脉注射放置。 24 G静脉导管放置在边缘耳静脉。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55193/55193fig2.jpg" /> 图2:小猪插管事件的顺序。 （ A ）将仔猪放置在侧卧位置进行插管。放置标准显示器。 （ B ）喉镜喉镜检查时，助手将舌头从口腔排出。 （ C ）会厌在形态上类似于人的会厌。在这个图形中，e刀片在vallecula。 （ D ）猪喉解剖与人类明显不同;声带距喉口几毫米深。在这个图形中，刀片尖端已经使会厌位移，露出喉。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55193/55193fig3.jpg" /> 图3:股动脉方法。 （ A ）使用强制空气加温装置维持动物温度。在整个程序中使用监控。用有色洗必泰制备广泛的无菌田，并用开窗无菌悬垂覆盖动物。 （ B ）腹股沟的触诊显示股动脉。 （ C ）大体上垂直于腹股沟皱纹并且长约1.5cm的阴茎皮肤切口被覆盖在股骨脉冲上。 （ D ）实现钝性解剖以暴露股骨神经血管束。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55193/55193fig4.jpg" /> 图4:股动脉插管。 （ A ）从外侧到内侧，神经血管束包含股神经，动脉和静脉。 （ B ）使用血管环和/或缝线分离股动脉。近端连接处的持续张力可防止动脉穿刺时出现过多的失血（见容器美白）。 （ C ）用针刺穿股骨a动脉，避免动脉后壁穿孔。当血液返回时，导丝通过针头进入动脉。 （ D ）取出针头，将导管推进导丝进入 股动脉。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55193/55193fig5.jpg" /> 图5:对照和异氟烷处理的仔猪的平均体重和年龄的比较。误差条表示+/- 1标准偏差，标准偏差值在每个误差栏上方。 <img alt="表格1" src="/files/ftp_upload/55193/55193table1.jpg" /> 表1:正常小猪生命体征总结，动脉al血气和血清电解质值与建议的修正方法。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193table1large.jpg" target="_blank">请点击这里查看此表的较大版本。</a> <img alt="表2" src="/files/ftp_upload/55193/55193table2.jpg" /> 表2:对照与异氟烷治疗的仔猪的平均体重和年龄的总结。进行不成对的双尾T检验，两组间差异无统计学意义。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193table2large.jpg" target="_blank">请点击这里查看此表的较大版本。</a> <img alt="表3" src="/files/ftp_upload/55193/55193table3.jpg" /> 表3:异氟烷治疗动物的平均生命体征和实验室价值。在过程中手术，生命体征和实验室值与正常范围的偏差及时纠正。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55193/55193table3large.jpg" target="_blank">请点击这里查看此表的较大版本。</a>

Watch this Scientific Journal Video about Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach at JoVE.com

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

麻醉诱导的发育神经毒性（AIDN）研究集中在啮齿动物，其不广泛适用于人类。非人灵长类动物模型更相关，但成本高昂，难以用于实验。相比之下，仔猪是一种临床相关的实用动物模型，是研究麻醉神经毒性的理想选择。

使用仔猪模型研究麻醉诱发的发育神经毒性（AIDN）:一种转化神经科学方法

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

use-piglet-model-for-study-anesthetic-induced-developmental

Nanyang Technological University

Research

JoVE Journal

Medicine

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

11.2K 次观看.  Ohio State University College of Medicine. 麻醉诱导的发育神经毒性（AIDN）研究集中在啮齿动物，其不广泛适用于人类。非人灵长类动物模型更相关，但成本高昂，难以用于实验。相比之下，仔猪是一种临床相关的实用动物模型，是研究麻醉神经毒性的理想选择。 

视频: Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

的的膀胱测压小老鼠:A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...