NS65819（CDJ），NS049720（CDJ），CA097257（CDJ），CIRM DR1 - 01426 我们要感谢技术援助拉奎尔桑托斯。

1。全身给药方法虽然药物的全身交付是不是最有效的方法，为实现在中枢神经系统的药物浓度高，全身交付方便，深受患者所接受。在这里，我们将展示适当的程序，三个经常使用的全身给药方法：静脉注射，腹腔注射，灌胃。 A.静脉注射（尾静脉注射） <ol><li>在程序开始之前，每只小鼠的体重应记录在案。与所有的临床前研究，应定期监测体重（每周两次或更大），以评估潜在的药物毒性。鼠标的体积体重不超过1％，应注射在一个单一的时间。例如，流体不超过悬液0.2ml应注射在20克鼠标。所有静脉注射液应通过适当的方法消毒。 </li><li>鼠标应使用加热垫或加热灯的尾静脉扩张5-10分钟的热身。如果使用烤灯，监视鼠标在任何时候，以避免热疗。 </li><li>鼠标被转移到一个装置，抑制鼠标，同时允许访问尾静脉（见视频）。在鼠标尾部有四个可见的船只上背部和侧面的船只的静脉和船只上的腹侧方动脉。要访问的尾静脉，鼠标的尾部是最远端点举行，90度旋转，因此，静脉朝上。注射部位用酒精棉签清洁和一个28克胰岛素注射器，斜面端插入静脉，（见视频）。如果放置在静脉针是正确的，应该自由移动，没有压力。压力甚至超过5-10秒，慢慢注入药物。如果尾巴上出现水泡，停止注射，因为这表明不再是在静脉针。 </li><li>注射后，注射部位轻轻推压，直到出血停止。这通常需要30-60秒。监视器的小鼠在注射后5-10分钟，以确保没有再出血。 </li></ol> B.腹腔注射<ol><li>开始前注射，药物应装入一个28克针注射器。确保有空间注射器注射前柱塞（例如，如果注射200μL，确保注射器容量为300μL或更大）。 </li><li>从笼中取出鼠标，它的尾巴和它放到特定的材质表面，使鼠标握的东西。一笼盖通常是足够的。允许鼠标伸展它的身体，然后使用你的非惯用手，掌握鼠标的背部皮肤，轻轻捏你的拇指和食指和中指（见视频）之间尽可能皮肤照顾。翻转鼠标，使鼠标的腹部朝上。如果鼠标可以随意移动它的头，松开握，然后再试一次，这样才能避免在注射咬伤。 </li><li>随着你的惯用手，拿起注射器和针插入一个30度角进入下鼠标左键象限（见视频）。按住鼠标稍微倒将有助于推动机关，远离注射部位。为了确保针在腹腔内的空间，拉回注射器推杆。如果任何液体或血液在注射器中出现，然后针在腹腔内的空间，应予删除。如果没有液体注射器吸气，然后注入注射器内容与均匀的压力超过1-5秒，然后释放鼠标。 </li><li>监视器的小鼠在注射后5-10分钟，以确保鼠标返回到正常活动水平。 </li></ol> C.灌胃<ol><li>开始注射之前，记录鼠标的重量。灌胃，可交付的最大音量是每公斤体重10毫升。例如，为20克鼠标的最大音量将200μL。试图注入更大的卷可能会导致回流，这将导致不完整的药物输送。管理卷大于上述表示如果有必要，多达三个剂量可能超过24小时的管理。 </li><li>为了避免刺破食道，重要的是要测量每只小鼠灌胃针的长度。按住鼠标的最后一根肋骨弯曲灌胃针18G球尖，然后标记在鼠标的头部使用永久性标记（见视频）提示长度。在灌胃，这个标志将被停止点鼠标口针插入。 </li><li>使用相同的把手为腹腔注射抑制小鼠。灌胃针插入嘴里，在舌头，ADVANCE通过咽针。过去的停止标志，不要插入针。针应进行顺利，没有任何压力（见视频）。如果您遇到的压力，停止并退出针头，以避免进入肺部注射液。 </li><li>针到位，压低柱塞超过1-5秒，然后取出针插入，这是在同一角度。显示器鼠标5-10分钟，密切关注的任何迹象，这可能表明，液体进入肺部的呼吸困难。 </li></ol> 2。本地传递急性对流增强交付 A.探头建设耐回流老鼠CED导管尚未投放市场。在这里，我们将演示一个Krauze等人（2005年Krauze）首次描述了一个方法，是从适应的套管施工方法。 <ul><li>套管三部分组成（ 图1）：直径100um的石英管，通过它infusate流动，为结构支撑的刚性金属针，和灵活的聚四氟乙烯管加载的infusate 。 </li><li>为了获得刚性的金属针，使用明火融化的塑料上Surflo静脉导管（24克钢丝），并用小镊子取出金属针。其余的导管可以被丢弃。切割长度的石英管（外径0.163毫米），比金属针，用单刃刀片。作用在氰基丙烯酸酯为基础的快速胶（如Krazy胶）小降硅管，照顾没有得到任何油管两端的胶水。在金属针插入石英管，并让干燥5分钟。 </li><li>一旦干燥，石英管应紧紧贴针。二氧化硅的两端应修剪，使石英管伸出针和3mm的石英管伸出从平坦的（见视频）指出为2mm。 </li><li>剪切特氟隆管20厘米长的一节。轧辊的金属针，在小降的胶粘剂，再照顾，不要让石英管两端的胶水，因为这会堵塞套管。针插入深度为1cm的特氟隆管。 1分钟，让干燥。使用胶枪，热熔胶应用金属针和聚四氟乙烯管（见视频）之间的联合下降。确保各方覆盖整个关节，让至少1小时的干燥。长达一个星期的套管可以预先在室温下储存。 </li></ul> B.输液过程<ul><li>为了准备手术区，所有表面应喷洒消毒剂，如2％洗必泰溶液。在手术过程中，应穿着无菌手术手套。表面再铺上吸水窗帘。在手术区应放在以下耗材： <ul><li>加热垫，以保持鼠标体温</li><li>两个小培养皿;一个含有3％的双氧水，和一个含有2％洗必泰</li><li>无菌纱布和棉签</li><li>无菌一次性手术刀（21号） </li><li>无菌22克针</li><li>鼠标立体框架</li><li>控制的速率注射泵</li><li>蒸压小鼠皮肤订书机，订书钉，起钉器</li></ul></li><li>要设置海关套管，附加1ml注射器特氟隆管，用塑料注射器适配器。套管应贴的立体框架，使其垂直于表面手术（见视频）。消毒套管，填充70％的乙醇1ml注射器和压低柱塞通过套管运行的乙醇。使用无菌生理盐水，重复这一过程，并检查左右的套管接头有无渗漏。消毒导管外，70％的乙醇擦拭轻轻擦拭。 </li><li>用无菌生理盐水填充套管和再拉回来，使注射器套管绘制一个小气泡进入。这个气泡会在导管生理盐水分开的infusate。然后，回程infusate（见视频）。简要运行导管连接到注射器注射泵泵和总理。 </li><li>稳重鼠标使用注射麻醉剂，并准备用一块无菌纱布擦拭数次（5-10秒）皮肤浸在2％洗必泰溶液。眼药膏应适用的鼠标，在手术过程中保持充足的水分。用消毒的手术刀，沿着头骨中心创建一个矢状切口，约1.5厘米长（见视频）。头骨表面，然后用棉签在3％的双氧水溶液浸泡清洗。小心避免鼠标的眼睛越来越双氧水。头骨的缝合线应体现在这一点：如果它们是不可见的，轻轻拭与FR的头骨ESH棉签在3％的双氧水溶液浸泡。 </li><li>确定前囟门（ 图2），然后测量2mm到的权利和1mm这种结构后找到输液部位。使用无菌22克针，轻轻扭针对头骨（见视频）在这一点上创建一个洞的头骨。避免强迫针向下对头骨。 </li><li>此时，鼠标应放置在立体框架，并开始接受吸入麻醉药（见视频）。管理处于较低的水平（1％）的麻醉，并仔细监视鼠标呼吸速率的变化，相应调整了麻醉。 </li><li>在骷髅洞的套管，然后降低3mm的颅骨表面下的位置。开始输液，使用下面的利率和期限： <table border="0"><tbody><tr><td> 0.1μL/分钟5分钟</td><td rowspan="5"><img src="/files/ftp_upload/1992/1992tablefigure.png" alt="总容积10 UL" /></td></tr><tr><td> 0.2μL/分钟5分钟</td></tr><tr><td> 0.5μL/分钟5分钟</td></tr><tr><td> 0.8微升/分钟7.5分钟</td></tr><tr><td>关闭1分钟</td></tr></tbody></table></li><li>在输液结束时，慢慢取出套管和棉签用3％过氧化氢的头骨。应用无菌骨蜡孔（见视频）。使用镊子，共同绘制皮肤在颅骨和主食关闭。丁丙诺啡应管理手术后的疼痛缓解。 </li><li>手术后监视鼠标，直到它恢复意识和流动。由于程序的长度，它可能需要鼠标一个小时重新获得充分的活动。在加热垫的鼠笼，在这段时间内，以避免体温过低，并没有房子与其他活跃小鼠恢复鼠标。 </li><li>手术后一周皮肤订书钉应删除。 </li></ul> C. 在活体成像 infusate荧光标记，可以成像，海关管理，并可以监测信号强度的变化，以及信号的位置。通常情况下，最好是等待2-3小时后，输液的形象，从而使鼠标恢复输液。 <ul><li>对于麻醉期间成像，使用吸入麻醉剂的低水平。鼠标的位置，背侧成像站，（例如，IVIS Lumina的，卡尺生命科学，阿拉米达，加州）。使用适当的过滤器设置的福陆公司是成像，获取的图像。对于海关，一个成功的输液应显示大部分的物质在大脑附近输液部位（ 图3）。 </li></ul> 3。代表性的成果缺乏不良反应的治疗管理是一个成功的注射的重要指标。例如，尾静脉注射后在外观上不应该有变化的尾巴（例如，大小，颜色）。一个气泡或水泡，尾静脉注射后表示皮下，而不是静脉注射，治疗交付。腹腔注射，腹部的皮肤上或变色的撞击可能表明皮下注射或损坏内部结构。在灌胃，用鼠标吃力，呼吸或咳嗽，可能表明，流体注入肺部，而不是进肚子里，。 对于海关，神经功能评估治疗成功的管理是非常重要的。可能已收到参展癫痫或偏瘫的鼠标，是不适当的输液。如果使用一种荧光infusate， 在活体成像，可用于评估成功的管理（ 图3） 。如果infusate是不是局部注射部位，输液没有成功。 <img src="/files/ftp_upload/1992/1992fig1.jpg" alt="图1" /> 图1：土木工程署的套管和手术设置。对流增强交付手术的基本要素设置显示。一个microinfusion泵（A）是连接到输液导管，（二，显示在顶部扩大）。一个立体的框架（C）是用来定位的探针。此映像不包括取暖​​，并在手术过程中使用的麻醉设备。 <img src="/files/ftp_upload/1992/1992fig2.jpg" alt="图2" /> 图2：鼠标颅骨缝合线。土木工程署输液部位（红星），可以找到确定的矢状面和冠状缝合线的交点（前囟门），然后测量2毫米横向和1mm的前囟后。 <img src="/files/ftp_upload/1992/1992fig3.jpg" alt="图3" /> 图3：成功的土木工程署的代表结果。李由土木工程署与远红光的荧光染料标记posomes注入老鼠大脑，并在体内和体外成像。一个成功的输液显示了本地化，无论是在体内（A） 和体外（二）输液部位的荧光信号。信号应无泄漏到对侧大脑半球局部注入半球。 <img src="/files/ftp_upload/1992/1992fig4.jpg" alt="图4" /> 图4：成鼠脑干成功土木工程署的形象代表。荧光标记的脂质体注入大鼠脑干。在大鼠中，尽可能多20μL注入。可以核实体外解剖脑成像大鼠颅骨和排除体内成像注射的深度，但正确的输液位置增加厚度。

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+of+a+topoisomerase+I+inhibitor+(nanoliposomal+topotecan)+and+a+topoisomerase+II+inhibitor+(pegylated+liposomal+doxorubicin)+in+intracranial+brain+tumor+xenografts.">Convection-enhanced delivery of a topoisomerase I inhibitor (nanoliposomal topotecan) and a topoisomerase II inhibitor (pegylated liposomal doxorubicin) in intracranial brain tumor xenografts.</a> Neuro Oncol. 9, 20-28 (2007).</li></ol>

这里展示的所有程序，由加州大学旧金山分校的机构动物护理和使用委员会的批准。

任何治疗药物的疗效的临床前评价必须考虑到代理的药理特性和利益的目标组织。虽然系统性的管理方法，一般是更轻便和​​患者的耐受性更好，血 - 脑屏障的选择性，往往需要地方交付用于治疗中枢神经系统疾病的治疗。在这里，我们已经证明大脑：海关直接配送的方法之一。本地传递到大脑的其他形式包括直接管理到脑脊液，瘤内推注，并脑室注射。对于这些方法，治疗扩散性是极为重要的，差扩散的限制，覆盖区域从注射部位（耆那教的1989年，1994年波波）几毫米。相比之下，土木工程署使用正压，以增加药物分布（1994年波波）的领域，同时还使治疗针对特定的神经解剖结构。在我们的实验室，我们已经成功地执行海关到脑干，以及为在啮齿类动物的尾壳（图 4 ）。 协会的能力，在啮齿类动物模型进行海关已成为越来越重要的最大限度地提高疗效，在治疗中枢神经系统疾病的各类时，越来越感兴趣。海关可用于提供各种代理人，包括纯化蛋白质，小分子药物，和病毒（吉尔2003年，德劲2003年，Szerlip 2007年）。与海关可以治疗的疾病范围包括中枢神经系统的癌症（山下2007年），以及神经退行性疾病，如帕金森氏症（2003年吉尔）。由于土木工程署研究的不断扩大，需要在海关管理治疗的活体成像，也同样增加。虽然荧光成像表明这里可能会无法通过人类头骨可见，用人合作造影剂灌注（迪金逊2008）等方法吸引兴趣的适用性评估监测中枢神经系统疾病患者infusates。

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<td>Fisher</td>
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<td>AKA &#x201C;Nolvasan&#x201D;</td>
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<td>20g Needle</td>
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<td>Becton Dickinson</td>
<td>305175</td>
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<td>28g Needle (with syringe)</td>
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<td>Fisher</td>
<td>22-004-270</td>
<td>AKA Insulin Syringe</td>
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<td>3% Hydrogen Peroxide</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>H312P-4</td>
<td>Store away from light</td>
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<td>Cotton Swabs</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>23-400-100</td>
<td>Autoclave before use</td>
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<td>Fisher</td>
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<td>Dunlap</td>
<td>HP950</td>
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<td>Fisher</td>
<td>14-841-20</td>
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<td>MD-1000, MD-1001</td>
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<td>Caliper Life Sciences</td>
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<td>1272830</td>
<td>AKA &#x201C;Akwa Tears&#x201D;</td>
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<td>Fits on Luer Lock Syringes</td>
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<td>2000020</td>
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<td>Stoelting</td>
<td>59020</td>
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<td>Stereotaxic Frame</td>
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<td>Stoelting</td>
<td>51725</td>
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<td>Teflon Tubing</td>
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<td>Upchurch Scientific</td>
<td>1520</td>
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<td>Xenogen Lumina</td>
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<td>Caliper Life Sciences</td>
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systemic and local drug delivery for treating diseases of the central nervous system in rodent models

彻底的中枢神经系统（CNS）的临床前试验的疗法包括在评估疗效时考虑的管理和代理生物分布的路线。两者之间的主要分类管理，本地与系统性，全身交付方法往往是首选由于易于管理。然而，全身交付可能会导致不理想的药物浓度在中枢神经系统实现，并导致错误结论，关于代理疗效。局部给药方法更具侵入性的，但可能是必要的，以达到治疗中枢神经系统的药物浓度。在这里，我们表现出适当的技术，为全身给药途径：静脉注射，腹腔注射，灌胃。此外，我们为本地传递到大脑的方法：对流增强交付（CED）。使用荧光标记的化合物在体内成像和适当的药品监督管理局核查。使用小鼠模型的方法，但是，可以很容易地使用大鼠适应。

Watch this Scientific Journal Video about Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models at JoVE.com

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

彻底在中枢神经系统的药物，临床前试验，往往涉及的评估和比较药物体内分布与管理的具体路线协会。在这里，三个系统性交付的常用方法（静脉，腹腔，口腔），以及为本地传递方法（对流增强交付）在小鼠体内证明。

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 ...

systemic-local-drug-delivery-for-treating-diseases-central-nervous

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

Pharmacotherapy of Psychosis and Mania

SCIENTISTS IN ACTION

30.2K 次观看.  University of California, San Francisco - UCSF. 彻底在中枢神经系统的药物，临床前试验，往往涉及的评估和比较药物体内分布与管理的具体路线协会。在这里，三个系统性交付的常用方法（静脉，腹腔，口腔），以及为本地传递方法（对流增强交付）在小鼠体内证明。

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

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, ...

科研

神经科学

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 ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

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 specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

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 ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

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 ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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 ...