我们想感谢柯特梅泽Isis制药有关ICV丸手术提供意见，以及Isis制药作为一个整体来提供我们的实验室与艾滋病服务。此外，我们要感谢凯里沙纳对本文的审阅。 TMM和SLD由NIH资助P50AG005681，K08NS074194，R01NS078398支持的。

下面的协议已在圣路易斯华盛顿大学的机构动物护理和使用委员会批准，并在符合国家机构使用实验动物的健康指引。如果这是您第一次进行手术，我们建议寻找在朱庇特的文章介绍啮齿动物手术开始前14。 艾滋病服务可以同时通过渗透泵输液和一个单一的ICV静脉注射到小鼠的中枢神经系统。正因为如此，下面详述的程序被分成两个分部。每一种方法的优点和缺点会感动在讨论部分。是为成年C57BL6和B6C3小鼠的协议中使用的坐标。在表1中，可以找到相应的大鼠坐标。 Alzet释药渗透泵 1。 Alzet释药渗透泵的制备<ol><li>放下无菌DRAPe和小心掉落以下物品，同时照顾到它不直接接触任何东西，以保持无菌:渗透泵（次），流调制器（S），1毫升无菌注射器（次），手术刀片，油管部分管= 5台泵（1件）。对于老鼠，有三个选项泵:14天，28天，42天（ 图2A）。 </li><li>摘帽并填写无菌15-20毫升生理盐水50毫升锥形管。可以添加多达6个组装泵每锥形管。 </li><li>填补p60的培养皿中，用100％乙醇。添加适当数量的导管乙醇。老鼠，导管长度是2.5毫米。 </li><li>过滤每个ASO到尽可能多的1.5毫升离心管中必要的。包括Eppendorf管中充满了0.9％无菌生理盐水填充管。 </li><li>一旦一切准备就绪，戴上无菌手套。请务必不要碰任何东西，是不是无菌的。更改必要手套。 </li><li>对于每个ASO被使用，填补1 ml注射器和loadpumps通过将针插入顶部的孔，慢慢填充，直到过量泄漏出来。继续，直到所有的泵都充满。 </li><li>摘帽流调制器，并插入泵。推流的调制器，几乎所有的方式中，留下一个3-5毫米的间隙。 </li><li>使用手术刀刀片，切管到每一块5等份。径的管件，填写用无菌生理盐水，用1 ml注射器，流调制器的顶部插入到管子的一端。然后从乙醇中的插管，并把它连接到管子的另一端。现在该泵完全组装。 </li><li>到无菌的50毫升锥形管将完全组装泵，吸用生理盐水。允许吸泵来吸收液体发起的药物输送到适当的温度，以及来。吸时间:14天泵:4-6小时，28天泵:40小时，42天泵:60小时。当完成组装泵，封顶的锥形管，在一个干净的地方37℃水浴中，直到手术。 </li></ol> 2。手术前程序<ol><li>抹一切用70％乙醇消毒区和放下无菌布，在桌面上和stereotax创建一个无菌区。重要的是要注意在整个手术无菌区。打开如下:珠灭菌器，加热垫，灯，数字读数，氧气/的异氟烷系统（ 图1）。 </li><li>如果做一个单一的手术或手术的一整批像我们经常做的:1对镊子，弯止血钳1，1对小剪刀，1对小弯钝的剪刀，1大鼠骨刀，将需要以下项目，1直止血钳，5-0尼龙缝合线，用70％乙醇的1锥形管，用95％乙醇的1个锥形管，1与碘锥形管，1与过氧化氢的锥形管，包装的无菌棉签，一瓶超胶水，1管抗生素软膏，管眼润滑剂，电动剃须刀1。 </li><li>两对剪刀，镊子，弯止血钳的尖端放置到珠灭菌，持续15秒。取出并放置在70％的乙醇。当执行一个以上的手术，重新消毒的仪器的提示在动物之间。 </li><li>关闭异氟醚4％和O 2至0.4升/分钟以及直接气体流室。我们有我们的异氟醚/氧气系统校准，每年至少有一个，以确保我们提供适量的麻醉。将鼠标室等待，直到呼吸明显放缓。取出鼠标和执行脚趾捏，以确保鼠标完全是无意识的。 </li><li>剃须头发肩膀在两眼之间的从顶部。如果鼠标开始清醒过来，将鼠标放回室，直到鼠标完全是无意识的，用脚趾捏确认。将麻醉的小鼠上stereotax和推在鼻子的鼻锥。要温柔，就像牙齿和骨骼脆弱。 </li><li>直接的气流stereotax保持鼠标麻醉。重要的是偶尔进行检查，以确保鼠标完全是无意识的，整个手术用脚趾捏。固定头部与耳酒吧。我们喜欢练级耳棒，虽然针对侧脑室，完全水平的头骨是没有必要的，因为大尺寸的心室。 </li><li>关闭异氟醚的水平下降至2.0％，进行维修。 DAB眼膏到每只眼睛。在视频中看到，奠定了无菌的悬垂性，使鼠标在只有基本的颈部和头部暴露。然后消毒手术区域开始在剃光区域的中心向外移动，先用棉签蘸95％乙醇，碘棉签蘸擦顶的头部和颈部的圆周运动。 </li><li>我们建议使用直肠探头反馈温度控制加热系统，以确保鼠标的温度保持在一个稳定的37°C。如果鼠标的温度降低手术过程中，手术后的恢复可能需要更长的时间和产生的低温可导致异常蛋白质的活性，包括过度磷酸化的微管相关蛋白tau 15。加热系统建立后，你现在准备开始手术。 </li></ol> 3。 Alzet释药渗透泵植入术<ol><li>在手术开始之前，穿上一双新的无菌手套。然后，使用镊子和小剪刀，做一个切口在皮肤上，从上述动物的头骨到最大的基地的脖子，在两眼之间的</li><li>以钝剪刀朝上的曲线滑动下面的皮肤于颈部背对着左后肢的基础。应该没有阻力。如果有，取出剪刀，然后再试一次。这就形成了渗透泵皮下口袋。 </li><li>无线PE干净的头骨用无菌棉签，棉签蘸双氧水，以提高前囟门。 </li><li>擦拭下来的50毫升锥形管，包含泵泵保持无菌。小心地将泵从锥形管，用弯止血钳，采取额外的照顾，不碰泵的两侧锥形管。握住底座，的泵用钳子和推流调制器的其余部分的方式与弯止血钳。 </li><li>握住泵流调制器和油管满足用弯止血钳。将泵插入在脖子底部的皮肤下，并向着左后肢将其推回，只要其将无阻力。要小心，不要让导管触摸任何东西。 </li><li>弯止血钳，抢套管顶部的凹槽符合底座。套管驱动器移动到的位置，并确保到位。 </li><li>插管的基础上，将落单的超级胶水。脓h的套管顶部到驱动器和位置，以使油管直背指出。 </li><li>导管尖端触摸前囟门上的数字显示为零的坐标。提高导管和1.1毫米横向移动到右侧，0.5毫米后（ 图2B）。保持皮肤的出路，用弯止血钳。 </li><li>驱动器通过头骨薄的金属导管，直到塑料套管基座牢固地压靠在头骨顶部。金属导管，可直接驱动通过头骨的小鼠由于相对颅骨薄。如果使用老鼠，在颅骨上钻一个孔，才降低了导管。 </li><li>任何皮肤拉远离颅骨胶水。套管/导管到位套管驱动程序，等待1-2分钟胶水完全干燥。 </li><li>练习时，以评估导管置入，注入20-40微升为2.5％FastGreen染料通过油管（ 图2C）。等待2-3分钟，perfuse的鼠标用1X PBS 0.03％肝素，消除大脑。切冠导管部位。如果导管放置在心室，染料灌注整个鼠标脑室系统（ 图2D）。 </li><li>保持导管的位置，用弯止血钳，同时提高驾驶员。慢慢松开止血钳套管以确保妥善地稳固​​的头骨。 </li><li>用棉签，按下顶部的套管。适合大鼠骨剪，到套管顶部和底部之间的小树林。剪掉，同时还用棉签按住顶部的套管。尽量保持快船水平不分离的套管从头骨。如果套管不脱胶，快速地重新胶水和施加压力，用棉签额外2分钟。 </li><li>用5-0丝线缝合，常规止血，镊子，缝合沿着全面开放，付出额外的关注，就在套管。我们使用一个飞奔g水平褥式缝合（ 见图5），由于头骨较大的切口。 </li><li>应用抗生素软膏，在头部和颈部轻拍。拧耳朵酒吧和松开的鼻锥。将鼠标恢复变暖垫从stereotax的地点。密切监测小鼠恢复的持续时间，一般为10-30分钟。每隔2-3分钟，直到鼠标开始走动和仪容产品本身。 </li></ol> 4。手术后的护理<ol><li>监测小鼠每天在手术后的第一周。 </li><li>如果鼠标出现疼痛或痛苦，我们建议提供鼠标与5毫克/公斤皮下注射卡洛芬每隔24小时至5天，以减轻疼痛。 </li><li>如果出现感染，慷慨地应用抗生素软膏，每日的区域，并咨询主治兽医，以确保正确的伤口愈合。 </li><li> ŗ5-0尼龙线的eMove后7-10天，以防止手术缝合线的刺激。 </li></ol> 5。更改或删除Alzet释药渗透泵泵后完成积极注入ASO，它可以改变或完全除去。 <ol><li>按照上述相同的预手术过程有以下变化:1）你将不需要的弯剪，弯止血钳，鼠骨刀，也没有数字显示，2），而不是跃跃欲试头的鼠标，刮胡子，的交界处的泵和管道，鼠标背面的消毒。 </li><li>鼠标的背面制作一个1.5厘米的切口，垂直于泵/油管结，使用镊子和剪刀。小心地拉出，泵出的切口无显著拉/推管，因为它可能会摇晃导管的。 </li><li>要卸下泵，夹管约0.5厘米以上的流量调制器和触摸强力胶油管吨Ø密封关闭。等待1-2分钟，胶水干。将回油管进入切口和缝合关闭。 DAB抗生素软膏到缝合皮肤和鼠标上加热复苏垫。 </li><li>改变水泵，新鲜制成的泵可充满95％的乙醇与10厘米的菜。导管并不需要与新泵。 </li><li>抓住一个新泵的镊子。成乙醇，沾你的手指，抓住泵，并丢弃流调制。然后，小心地，慢慢地拉过的旧泵流调制器连接到油管和套管。只要它是慢慢滑下新泵，确保流调制器不会接触到外面的鼠标。 </li><li>重新插入新的泵通过切口进入皮下包和缝合皮肤。 DAB抗生素软膏上的伤口和地点恢复鼠标垫。 </li><li>正如泵注入，监测小鼠手术后每天检查为p泉/不适和感染。卡洛芬和抗生素治疗，如认为有必要。 </li></ol> ICV六味地黄丸 6。手术前程序<ol><li>按照上述第2相同的预手术过程有以下变化:1）你将不需要弯剪，弯止血钳，或大鼠骨刀; 2）清洗10微升汉密尔顿注射器和针头用无菌水和负载ASO;和3）将位于套管驱动程序的立体定位仪上的注射器和针头。 </li></ol> 7。静脉注射ASO <ol><li>使用镊子和小剪刀，做一个切口从基地的脖子，眼睛之间的。 </li><li>擦拭干净，用无菌棉签，然后用棉签蘸双氧水，以提高前囟门的头骨。 </li><li>将注射器/针到位和安全。确保针斜面部分面临后路。降低针，直到它触及前囟门。零点的坐标。移动针横向右1.0毫米，前0.3毫米（ 图3A）。 </li><li>缓慢驾驶针通过头骨，直到针孔与颅骨的顶部平齐。再次，这是可以做到由于鼠标颅骨的薄。归零的z坐标和，降低针-3.0毫米的速度为1毫米/秒（ 图3B）。等待2-3分钟，大脑密封针的周围。 </li><li>在1微升每秒的速率，提供完整的10微升的ASO。等待2-3分钟。艾滋病服务入脑室，我们使用1微升输液速度，这个速度虽然在不同药物之间可能会有所不同。练习时，检查针妥善安置提供为2.5％FastGreen染料10-20微升。用1X PBS 0.03％肝素灌注小鼠染料输液后不久，使大脑冠状切片，以验证该染料是在脑室系统（ 图3C）。 </li> &lt;LI&gt;握住棉签对头骨在基地的针。提高针每秒1毫米的速度。只要针的头骨，滚动棉签针孔，并保持1分钟，以防止任何泄漏ASO。 </li><li>缝合皮肤封闭，应用抗生素软膏，和地方加热复苏鼠标垫。它根据ASO集中，可能需要20分钟到几个小时才能完全恢复。 </li><li>正如与泵注入，监测小鼠手术后每天检查疼痛/不适和感染。卡洛芬和抗生素治疗，如认为有必要。 </li></ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+oligonucleotide+therapeutics+for+inherited+neurodegenerative+diseases.">Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases.</a> Trends in Molecular Medicine. , (2012).</li><li>Kole, R., Krainer, A. R., Altman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+therapeutics:+beyond+RNA+interference+and+antisense+oligonucleotides.">RNA therapeutics: beyond RNA interference and antisense oligonucleotides.</a> Nature Reviews Drug Discovery. 11, 125-140 (2012).</li></ol>

作者从Isis制药收到他们的反义寡核苷酸。

有能力提供全球药物在中枢神经系统，在视频中显示的是一个非常强大的技术，很容易学习和使用。与实践中，一个单泵植入术或ICV丸的可以在10分钟内完成，允许在同一时间被视为大老鼠同伙。这是特别有用的研究与行为读出，随着大量的鼠标行为是至关重要的，帮忙看看显著差异。 根据我们的经验， 通过泵和注射丸提供艾滋病服务，我们已经观察到一些优点和缺点，每个方法。应当指出，这些都是在我们的实验室的意见，可能不是真的，所有的小鼠和大鼠模型。 我们发现，使用泵的优点是他们有能力提供高量分布在更长的一段时间，因为ASO的ASO。这通常等同于较长的持续击倒或拼接后活跃ASO输液，虽然这可能并不总是如此。泵也允许精确的时间框架阿苏交货（14天，28天或42天），并允许甚至更长的时间积极ASO输液泵可以更改一次。但是，我们已经注意到，改变泵多于一次增加了可变性，由于防止泵的泵，从适当的吸收液体的纤维周围的口袋形成。泵的缺点是，一些老鼠不容忍泵，以及其他。如果您正在使用的是转基因株系比较脆弱，泵可能是太麻烦了。该泵还需要被删除的最终输注后，对小鼠其他手术和增值麻醉。如果做行为的工作，这是特别重要的是消除泵，让泵存在的至少1-2周的恢复时间会影响在小鼠体内的某些行为。 与ICV丸注射，一个优点是成本。有一个前期投资购买的注射器和针头，但随着时间的推移，静推注射更具成本效益，因为没有泵/管材/导管购买。总体来说，保持侧脑室注射丸由于缺乏套管和水泵。我们还发现，ICV丸可以用来提供艾滋病服务年轻的和/或更脆弱的小鼠。 ICV丸剂的一个缺点是，它是单次注射。没有那么多整体ASO可以通过这条线路传送，如果所使用的ASO的作用的持续时间很短，拦截/剪接的影响也将是短暂的。 无论是渗透泵以及ICV丸剂有能力提供艾滋病服务，可以拦截蛋白质或改变基因的剪接在各处的啮齿动物中枢神经系统中，一种技术，神经科学相关的多个领域中有着广泛的应用。我们建议试点交付这两种方法，如果你不确定哪种给药途径最适合您的S研究pecific。

有些药物是无法穿越血脑屏障（BBB），要求直接中枢神经系统（CNS）交付。要绕过血脑屏障，药物可直接传送到大脑使用所描述的方法。而我们的实验室和文件中有详细的本发明专注于反义寡核苷酸（艾滋病服务），其他药物，如小分子，抗体，基因治疗的载体， 等等 ，也可以被传递通过相同的方法。 某些蛋白质在神经退行性疾病的发病机制中发挥重要作用。这种蛋白质往往形成有毒的物种，并积累成聚集，导致最终的神经元死亡和随后的神经系统疾病3-4。在努力减缓甚至停止对这些疾病的进展，其中一个治疗方案可能是直接瞄准和减少致病蛋白质。然而，这些蛋白质经常发现无所不在通过中枢神经系统，使其难以电子<html> ffectively他们的目标在全球范围内。 为了在整个中枢神经系统的靶基因，我们管理艾滋病服务到小鼠脑脊液（CSF） 通过侧脑室绕过血脑屏障。这种特殊的方法充分利用沐浴整个大脑和脊髓的鼠标脑室系统，使广泛分布的ASOS。我们使用18-20聚体的RNA分子直接结合靶mRNA序列的艾滋病服务，并根据对ASO的化学修饰，无论是A）招募的RNase-H的mRNA降解导致击倒或B）的选择性剪接1转移2,16,17。应当指出的是，存在多个分子撞倒一个特定的蛋白质在体内 ，包括shRNA的。由于这些分子是不是本文的重点，我们引导读者阅读的文章，更好的细节击倒作用机制和各5-6的优势/劣势。 jove_content"&gt;在之前的工作中，我们已经使用了艾滋病服务目标蛋白在肌萎缩性脊髓侧索硬化症（ALS）7（ 图4）的转基因大鼠模型中的超氧化物歧化酶（SOD1）SOD1突变发生在约2％的所有ALS例8，虽然它已被最近虚拟SOD1可以发挥重要的作用，以及9-10。在散发性ALS的发病后显着增加通过减少总SOD1水平ALS转基因大鼠，生存。这些重要的数据成为第一个表明，在中枢神经系统中可能有ASO治疗神经疾病模式的深远而积极的影响。针对人类SOD1从那时起，艾滋病已经进入，并成功地完成了I期人体临床试验，以最小的副作用（Clnicaltrails.gov NCT01041222） ，提出在2012年第 64届美国神经病学学会计划向前移动的ASOS II期临床试验目前正在进行中。 <p类=的"jove_content"&gt;虽然定位SOD1是第一次使用一种神经系统疾病治疗的艾滋病服务示范，其他几个研究至今已进行看着不同的疾病及彼等各自的蛋白质目标。在2010年和2011年，艾滋病，移位拼接的蛋白质运动神经元生存2（SMN2）被用来在脊髓肌肉萎缩症（SMA）的转基因小鼠模型，并在显着改善疾病表型11,12。现在，这些拼接ASOS在第一阶段我临床试验SMA患儿（NCT01494701 Clinicaltrails.gov）。此外，近期瞬态针对亨廷顿基因的艾滋病服务管理可以显着地抢救亨廷顿的小鼠模型，即使在亨廷顿蛋白水平恢复到基线13。 在所有这些研究中，艾滋病被送到侧脑室，以减少总的基因水平或通过改变基因拼接整个中枢神经系统。可用于这两种渗透泵和一个单一的大剂量注射提供艾滋病服务的CSF。泵允许一个缓慢的，持续的交付，，而侧脑室丸是一种快速，一次性注射。我们都用了这些方法的成功，虽然我们还没有报道泵和丸在一个单一的转基因株系之间的直接比较。 在中枢神经系统中使用艾滋病服务是一个功能强大的方式，以减少总蛋白水平和/或改变的几种蛋白质的拼接。虽然我们使用的艾滋病服务专为治疗神经系统疾病，我们认识到，其他领域也可能受益于这种技术。感兴趣的蛋白质只要是在中枢神经系统中表达和最终目标是实现中枢神经系统范围内的基因表达的变化，使用艾滋病服务中所证明的技术可能是非常有用的。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>PREPARING ALZET OSMOTIC PUMPS </td>
 </tr>
 <tr>
 <td>Alzet Osmotic Pump 14 days</td>
 <td>DURECT</td>
 <td>Model 1002</td>
 <td></td>
 </tr>
 <tr>
 <td>Alzet Osmotic Pump 28 days</td>
 <td>DURECT</td>
 <td>Model 2004</td>
 <td></td>
 </tr>
 <tr>
 <td>Alzet Osmotic Pump 42 days</td>
 <td>DURECT</td>
 <td>Model 2006</td>
 <td></td>
 </tr>
 <tr>
 <td>2.5 mm Catheters</td>
 <td>PlasticsOne</td>
 <td>3280PM/SPC</td>
 <td>Custom ordered to 2.5 mm Catheter Length</td>
 </tr>
 <tr>
 <td>Vinyl Catheter Tubing</td>
 <td>DURECT</td>
 <td>7760</td>
 <td>ID: 0.027", OD: 0.045"</td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride, Irrigation, USP</td>
 <td>Baxter</td>
 <td>2F7124</td>
 <td>NOT to be used in pumps or tubing</td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride, Injection, USP</td>
 <td>Hospira</td>
 <td>NDC 0409-4888-10</td>
 <td></td>
 </tr>
 <tr>
 <td>p60 Petri Dish (Sterilized)</td>
 <td>TRP</td>
 <td>93060</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical Blades (Sterile)</td>
 <td>Butler Schein</td>
 <td>#007319</td>
 <td></td>
 </tr>
 <tr>
 <td>Latex Surgical Gloves (Sterile)</td>
 <td>Micro-Touch</td>
 <td></td>
 <td>CatNo will depend on size of the gloves needed</td>
 </tr>
 <tr>
 <td>Sterile Towel Drape</td>
 <td>Dynarex</td>
 <td>4410</td>
 <td></td>
 </tr>
 <tr>
 <td>.2um Syringe Filters</td>
 <td>PALL</td>
 <td>4192</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml Syringe (Sterile)</td>
 <td>BD</td>
 <td>309625</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml Conical Tubes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>100% Ethanol</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>PUMP &amp; BOLUS SURGERY PROTOCOLS </td>
 </tr>
 <tr>
 <td>Curved Forceps</td>
 <td>Fine Science Tools</td>
 <td>11001-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Curved Hemostat</td>
 <td>Fine Science Tools</td>
 <td>13009-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine Sharp Scissors</td>
 <td>Fine Science Tools</td>
 <td>14060-09</td>
 <td></td>
 </tr>
 <tr>
 <td>Curved Blunt Scissors</td>
 <td>Fine Science Tools</td>
 <td>14029-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Bone Cutter</td>
 <td>Fine Science Tools</td>
 <td>16104-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Straight Hemostat</td>
 <td>Fine Science Tools</td>
 <td>12002-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>7653-01</td>
 <td>10 &mu;l gas-tight with removable needles</td>
 </tr>
 <tr>
 <td>Needles</td>
 <td>Hamilton</td>
 <td>7758-04</td>
 <td>26 gauge, Point Style: 2</td>
 </tr>
 <tr>
 <td>5-0 Nylon Suture Thread</td>
 <td>Covidient</td>
 <td>SN-871</td>
 <td></td>
 </tr>
 <tr>
 <td>Alcohol Pads</td>
 <td>Select</td>
 <td>#521</td>
 <td></td>
 </tr>
 <tr>
 <td>Cotton Swabs (sterile)</td>
 <td>Puritan</td>
 <td>REF 806-WC</td>
 <td></td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Loctite</td>
 <td></td>
 <td>Longneck Bottles</td>
 </tr>
 <tr>
 <td>CAUTION: FastGreen Dye</td>
 <td>Sigma</td>
 <td>F7252-5G</td>
 <td>Wear Eyeshields and Gloves when handling this product</td>
 </tr>
 <tr>
 <td>Antibiotic Cream</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Eye Ointment</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Electric Shaver</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>70% Ethanol</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>10% Provadone Iodine</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>3% Hydogen Peroxide</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Warming Pad</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Bead Sterilizer</td>
 <td>SouthPointe Surgical</td>
 <td>GRM5-1450</td>
 <td></td>
 </tr>
 <tr>
 <td>Small Animal Stereotaxic</td>
 <td>Kopf</td>
 <td>Model 940</td>
 <td></td>
 </tr>
 <tr>
 <td>Nose Cone</td>
 <td>Kopf</td>
 <td>Model 923-B</td>
 <td></td>
 </tr>
 <tr>
 <td>Ear Bars</td>
 <td>Kopf</td>
 <td>Model 921</td>
 <td>This model is optional</td>
 </tr>
 <tr>
 <td>Cannula Driver</td>
 <td>Kopf</td>
 <td>Model 1966</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe Holder</td>
 <td>Kopf</td>
 <td>Model 1972</td>
 <td></td>
 </tr>
 <tr>
 <td>Temperature Control System</td>
 <td>Kopf</td>
 <td>Model TCAT-2LV</td>
 <td>Optional</td>
 </tr>
 <tr>
 <td>Oxygen/Isoflurane System</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

direct intraventricular delivery of drugs to the rodent central nervous system

由于不能穿过血脑屏障，某些药物需要直接传递到中枢神经系统（CNS）。我们的实验室，专门集中于反义寡核苷酸（ASOS），虽然在这里的视频显示技术也可以用来传送了大量的其他药物的中枢神经系统。反义寡核苷酸（艾滋病服务）具有序列特异性敲除目标1以及特定基因的2的移位异构体比率的能力。为了实现广泛的基因敲除小鼠在中枢神经系统中的拼接，艾滋病服务可交付使用两个单独的给药途径，这两者中，我们证明在视频进入大脑。 第一个使用Alzet渗透泵，连接到外科手术植入的导管入侧脑室。这允许进入中枢神经系统在指定的一段时间内连续输注的艾滋病服务。第二个涉及到一个单一的静脉注射一喜GH浓度的ASO入右侧脑室。两种方法都使用鼠标脑室系统提供整个大脑和脊髓的ASO，虽然根据研究的需要，一种方法可能会比其他优选的。

泵输注后ICV推注后，或在指定的时间（我们经常使用四个星期），它是重要的ASOS测试的有效性。我们建议采取来衡量各地区的脑和脊髓的水平/亚型的靶基因，采用实时定量PCR在mRNA水平以及蛋白质水平，可以使用免疫印迹或ELISA已发布的例子（ 图4 ）。这将有助于确定整个中枢神经系统不同区域ASO疗效。如果目标蛋白的半衰期很长，我们建议等待更长的ASO输液后，以评估最大的蛋白质击倒或拼接。 同样重要的是，以测试寡核苷酸分布。泵输注导致同侧和对侧半球的分布通过，但ASO浓度较高的地区是经常出现在接近脑室系统16。 ICV静脉推注注射产生更多的unifo，RM寡分布通过中枢神经系统的16，但效果可能不会持续很长时间。我们引导读者南威尔斯马等。泵与丸寡分布16看到一个直接的比较。 我们还建议测试多个剂量的持续时间以及艾滋病服务的行动，因为每个ASO将体内的行为略有不同。艾滋病服务通常有一个相对较长的作用时间，与目标击倒或拼接持续几个月后ASO交付的。此外，一些艾滋病服务会比别人更好一些的靶基因会比别人更容易拦截。正因为如此，当筛选ASOS，以确定引线的候选，我们推荐的测试至少3-5艾滋病服务在体内有n = 4-6成年小鼠每ASO作为第一通。 如果使用一种药物艾滋病服务以外，它也将是重要的导频了理想的药物浓度，药物的作用持续时间，和中枢神经系统的药物该特定化合物的分布。 <img alt="表1" fo:content-width="5in" fo:src="/files/ftp_upload/50326/50326table1highres.jpg" src="/files/ftp_upload/50326/50326table1.jpg" /> 表1中。外科坐标的坐标用来打右侧脑室在小鼠和大鼠植入渗透泵时，以及进行侧脑室丸。两个坐标，的泵和ICV丸，右侧脑室目标。我们使用了两个不同的组，因为这些都是我们所拥有的最丰富的经验和知道ASO提供完善的经销。 <img alt="图1" src="/files/ftp_upload/50326/50326fig1.jpg" /> 图1。立体定位设置。 （一）必要的立体设备Alzet释药泵和丸注射手术。）玻璃珠消毒（C）河2）数字读数。3）立体定向移动武器基地。 四）温度控制加热垫。V）异氟醚/氧气系统。 六）异氟醚商会（B）特写鼻锥和耳棒。两个附件。左:注射器/持针为ICV丸注射。右:渗透泵植入术插管驱动。 <img alt="图2" src="/files/ftp_upload/50326/50326fig2.jpg" /> 图2。 Alzet渗透泵注入。 （A）ALZET渗透泵选项小鼠。 14天泵（0.25微升/小时），28天泵（0.25微升/小时），42天泵（0.15微升/小时）（B）坐标为前囟门泵植入后，-0.5毫米-1.1毫米横向（右），（C）-2.5毫米腹（导管长度）。驱动导管通过后头骨和胶合所述套管，染料通过导管冲洗评估妥善安置在导管在侧脑室（D）后立即灌注脑染料（C）中，给药。如果成功地放置在脑室导管，染料将分布在整个鼠标脑室系统。 <img alt="图3" src="/files/ftp_upload/50326/50326fig3.jpg" /> 图3。侧脑室注射。 （A）坐标为前囟门推注:+0.3毫米，-1.0毫米横向（右），-3.0毫米腹前（B）驾驶通过头骨-3.0毫米腹针后，染料通过注射器推评估针妥善安置在购买心室（C）（B）中的染料给药后不久灌注脑。如果针被成功放置在心室，染料将被分布在整个鼠标脑室系统。 <img alt="图4" fo:content-width="5.5in" fo:src="/files/ftp_upload/50326/50326fig4highres.jpg" src="/files/ftp_upload/50326/50326fig4.jpg" /> 图4。反义寡核苷酸降低大鼠体内的SOD1（图直接取自Smith 等人7）。 （AD）的 SOD1的反义寡核苷酸SOD r146192或超氧化物歧化酶（SOD）被注入炒右侧脑室正常大鼠28天到100微克/天（A）大脑和脊髓内源性SOD1 mRNA水平作为衡量定量RT-PCR（ B）SOD1-微管蛋白水平ASO输液后（C和D）蛋白质水平的微管蛋白和SOD1量化脑内灌注不同地区艾滋病服务（E）对素1或GSK-3的公测2周被注入到非转基因小鼠和mRNA水平评估在右侧额叶/颞叶皮层中右侧脑室。从美国临床研究学会的许可。转载<a href="https://www.jove.com/files/ftp_upload/50326/50326fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/50326/50326fig5.jpg" fo:content-width="3in" fo:src="/files/ftp_upload/50326/50326fig5highres.jpg"/> 图5。运行水平褥式缝合。5-0尼龙缝合线和编织的皮肤，开始前和工作后，更多地集中在套管。一旦达到最后点，缝合线，然后带回并拧在套管一次。这将确保适当的伤口闭合，防止套管/油管通过皮肤工作。

Watch this Scientific Journal Video about Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System at JoVE.com

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

我们描述了一种方法，目标通过植入导管或进行大剂量注射到在小鼠右侧脑室中枢神经系统的药物。我们特别专注于交付反义寡核苷酸。这种技术是很容易适应其他药物和大鼠。

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

direct-intraventricular-delivery-drugs-to-rodent-central-nervous

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

Pharmacotherapy of Psychosis and Mania

SCIENTISTS IN ACTION

59.9K 次观看.  Washington University in St. Louis School of Medicine. 我们描述了一种方法，目标通过植入导管或进行大剂量注射到在小鼠右侧脑室中枢神经系统的药物。我们特别专注于交付反义寡核苷酸。这种技术是很容易适应其他药物和大鼠。

视频: Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for further development of new compounds. A computerized and thoroughly validated battery of neuropsychological and neurophysiological tests has been shown to be sensitive to detect drug-induced effects of multiple new and existing compounds. The test battery covers the main CNS domains, which have been shown to respond to drug effects and can be repeatedly administered following drug administration to characterize the concentration-effect profile of a drug.
The standard tests in the battery are saccadic eye movement, smooth pursuit eye movement, the Bowdle visual analog scale (VAS), the Bond and Lader VAS, body sway, adaptive tracking, visual verbal learning, and quantitative electroencephalography (qEEG). However, the test battery is adaptive in nature, meaning that it can be composed and adjusted with tests fit to investigate specific drug classes, or even specific receptors.
Showing effects of new cholinergic drugs designed to have a pro-cognitive outcome has been difficult. The pharmacological challenge model is a tool for early proof-of-pharmacology. Here, a marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. The test battery was implemented to investigate the potential of the nicotinic receptor antagonist mecamylamine to be used as a challenge model for cholinergic dysfunction, as seen in neurodegenerative disorders.
A worsening of scores in a dose dependent manner on the visual verbal learning test (VVLT; a test for learning and memory abilities) and the adaptive tracking test (a measure of visuomotor control and arousal), in particular, showed that the test battery is sensitive to showing acute pharmacodynamic effect after administration of anti-cholinergic drugs.

A validated computerized battery of neuropsychological and neurophysiological tests is used to study pharmacodynamic effects on the central nervous system of newly developed drugs in early phase development. To demonstrate the test battery, the acute effects of mecamylamine and the reversal of these effects by two agonist drugs are described.

一种研究胆碱能药物早期开发中胆碱能药物中央神经系统药效学作用的计算机测试电池

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

啮齿动物的中枢神经系统药物直接脑室交货

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