This work was supported by the Dr. Werner Jackst&#228;dt Foundation (to Eduardo Sanchez-Mendoza), the German Academic Exchange Service (DAAD; to Jeismar Carballo), the German Research Council (HE3173/2-1, HE3173/2-2, and HE3173/3-1; to Dirk M. Hermann), Heinz Nixdorf Foundation (to Dirk M. Hermann).

动物实验进行政府审批（G1361 / 13，AZ84-02.04.2013.A192和G1362 / 13，AZ84-02.04.2013.A194; Bezirksregierung杜塞尔多夫）的基础上对实验动物的护理和使用美国国立卫生研究院的指导方针。 泵1.准备Miniosmotic的<ol><li>在无菌环境中（即，细胞培养罩），获得泵，导管，流调节剂，脑输注插管和间隔盘被使用（图1）。 </li><li>用于递送药物直接入脑室使用一个间隔件光盘，从而使插管针头的仅前端与所述心室接触。握住套管用钳子倒挂。加入两滴氰基丙烯酸酯粘合剂（CA），并介绍了一个间隔盘。 </li><li>离开套管上朝上的平坦表面，使得衬垫圆盘和套管变干之间的粘合剂。 </li><li>切导管在2.5厘米左右的部分。 </li><li>对于EA通道泵，准备一个最小300-350微升感兴趣的溶液中的，因为这将需要以完全填充从而挤压所有空气泵在其内部，以防止在导管内形成气泡。 气泡会阻碍的流溶液进入大脑。 </li><li>小心地将导管连接至流量主持人。 </li><li>使用2毫升注射器连接到由脑输注套件中提供的针，填充泵直至溶液少量逃逸的泵，因此防止它里面的气泡。 </li><li>仔细填写流动主持人和导管注意防止残留在导管内有气泡。 </li><li>引入泵内的流动调节剂。 </li><li>一旦填充，小心地将插管连接至所述导管的末端。如果观察到气泡形成取出插管，并仔细再填充导管载体或药物溶液，然后重新引入插管。 </li><li>放置PU熔点在用无菌生理盐水溶液的容器和离开它在37℃CO / N。 </li><li>植入前重新检查气泡形成。如果需要的话（ 即 ，气泡观察）取下套管和填充管。重新连接套管。这应该采取的解决方案只有几微升。 </li></ol> 2.植入Miniosmotic泵注:对于这些实验动物用1％异氟醚（30％O 2，70％N 2 O）麻醉。然而，如果不能使用这种办法，使用麻醉的腹膜内注射也是可能的11。 <ol><li>定位在麻醉下的立体定向装置中的动物并覆盖眼睛的保护性软膏，以防止干燥时在麻醉下。通过当用手指或用钳子按压观察缺乏响应在后爪的确认麻醉。不要继续，直到动物是完全睡着了。 </li><li>切皮毛在他广告无论是用剪刀或剃齿机。切尽可能不损伤皮肤。 </li><li>清洁用70％乙醇和消毒剂具有抗菌和杀真菌剂性质的皮肤。 </li><li>用手术刀略微打开1厘米的切口中线的右和暴露颅骨。 </li><li>清洁用棉签头骨。用70％乙醇浸泡，并通过它在头骨。这将导致颅骨干燥。 </li><li>如果头骨有轻微出血，使用cauterizer以消除任何出血点。血液可以防止在CA从干燥正确在骨一旦插管植入。 </li><li>使用立体定向装置，使上，其中所述套管将植入颅骨的点的标记。对左半球坐标心室输注是-0.2毫米尾椎，0.9毫米外侧前囟（图2A）。 </li><li>轻轻地钻在做一个45度角的头骨;这防止了钻意外进入的和腹腔ñ。反复演练了几秒钟，然后检查有多深的洞。停钻，一旦颅骨变薄，但还没有完全渗透。 </li><li>打破脑膜用无菌针的前端，直到完全进入大脑的实现。这样做轻轻以便不损伤大脑。 </li><li>清洁带用棉棒70％的乙醇的头骨。 </li><li>引进动物在安特罗尾方向在皮肤之下直镊子。使用镊子在所述泵将植入动物的背部皮肤下打开的空间。引入泵入动物离开导管和插管外（图2B）的背面。泵将保持在这个位置，直到它被删除，也不需要任何类型的固定。 </li><li>小心地将胶水四小滴旁边的套管针。 </li><li>仔细介绍通过头骨针无侧身移动它。保持在位置15-30秒üNTIL将插管完全附着（图2C.1）。一旦到位，所述插管将达到2.5毫米的dorso腹侧方向如果一个间隔盘已被使用。如果正确连接，插管将保持附着在骨，直到实验结束。 </li><li>将一个手指在移动选项卡，然后用另一只手按领口与一组剪刀（ 图2C.2）削减其关闭。 </li><li>闭合伤口在皮肤上用5-0缝合线，并添加几滴在伤口之上的聚维酮碘溶液（PVP-I），以防止感染（图2D）。 </li><li>移动动物到一个新的笼子。不要将手术动物与那些仍然没有被操作的动物。记住，已经被植入了在药物施用的时间独自在笼子里的动物的泵。 </li><li>不要让老鼠无人看管，直到他们清醒并恢复了胸骨斜卧。 </li></ol> 3.泵Remova升注意:通常在实验结束时的由泵允许的交货时间结束时，但它是可以除去的泵，以做二次实验作为后续于药物递送。为了做到道示踪剂注射它因此，有必要卸下泵。 <ol><li>放置在麻醉下seterotactic设备动物并覆盖眼睛保护软膏，以防止干燥时在麻醉下。通过当用手指或用钳子按压观察缺乏响应在后爪的确认麻醉。不要继续，直到动物是完全睡着了。 </li><li>小心通过在泵植入天所做的切口切削打开皮肤。 </li><li>用手术钳握住套管，将其拉出。它应该从头骨容易地除去。温柔出血预计，通过将棉签停止，等待1-2分钟。 </li><li>拉动泵出通过导管。他lp的它出来把它推开，产生压力在皮肤上。 </li><li>用5-0缝合线再次关闭伤口，并加入几滴PVP-I的。 </li><li>移动动物到一个新的笼子。不要把手术动物与那些仍然没有被操作的动物。已接受手术的动物可以放在一起。 </li><li>不要让老鼠无人看管，直到他们清醒并恢复了胸骨斜卧。 </li><li>让动物进入到道示踪剂注入之前恢复10天。 </li></ol> 4.压力道示踪剂注入以45°角上的右运动皮层<ol><li>放置在麻醉下的立体定向设备上的动物并覆盖眼睛的保护性软膏，以防止干燥时在麻醉下。通过当用手指或用钳子按压观察缺乏响应在后爪的确认麻醉。不要继续，直到动物是完全睡着了。 </li><li>打开头上的伤口。 </li><li> C瘦的头骨用70％乙醇棉签。 </li><li>关于该装置的垂直支架引入一个5微升玻璃注射器（26Sガ）。确保玻璃保持器的下端正好在夹持片。注射器一定不能过远低于它作为那么这将是不可能将其放置在45℃从垂直位置并进行注射。 </li><li>直接东方注射器超过前囟门，然后找到所需的坐标。对于注射液对运动皮层坐标:点＃1:0.5毫米吻端至2.5mm横向相对于前囟门。点＃2:1.34毫米喙，2.5毫米横向相对于前囟门。 </li><li>一旦坐标已经被找到，表明其在与细尖标记头骨位置。进行一次完整的头骨过度墨水释放可能会隐藏正确的坐标点。 </li><li>小心钻颅骨拿着电钻在45℃。如步骤2.8，经常检​​查头骨，使其不能完全PERForated为了避免损坏所造成的钻头大脑。使用注射器的尖端，以确保所有的骨已被删除。 </li><li>装载600 NL的道示踪稀释到注射器。 </li><li>调整向右侧的动物，以45°的垂直位置。在显微镜下，将注射器的针的尖端正好在孔的前面。 </li><li>通过1.5毫米移动它在垂直方向上。让针稳定在这个姿势30秒至1分钟做压力注射前。 </li><li>注入300 NL示踪稀释的三个步骤100 NL由30秒从彼此分离。在最后一次注射之后，离开注射器稳定30秒到1分钟，以避免所述示踪剂流出的大脑。 </li><li>慢慢拉出注射器，重新定位在第二孔并用剩余300 NL属于注射器内重复相同的过程。 </li><li>第二次注射后，取出注射器并继续关闭WOU第二用5-0缝合加几滴PVP-I的。 </li><li>移动动物到一个新的笼子。不要把手术动物与那些仍然没有被操作的动物。已接受手术的动物可以放在一起。不要让老鼠无人看管，直到他们清醒并恢复了胸骨斜卧。 </li><li>让动物恢复牺牲前10天。 </li></ol> 5.道示踪观察<ol><li>每个机构的指导方针完全麻醉的动物。通过当用手指或用钳子按压观察缺乏响应在后爪的确认麻醉。不要继续，直到动物是完全睡着了。 </li><li>灌注动物用PBS中的4％多聚甲醛（pH为7）根据标准方案6。 </li><li>通过O / N浸泡在4％多聚甲醛消除大脑和后固定。 </li><li> Cryoprotect蔗糖的大脑在30％左右，直到大脑下沉。然后取出大脑和10秒imme其冻结rsion在液氮中。保持大脑在-80℃直至部分产生的。 </li><li>通过切片上cryostate生产的20微米的部分逆行道追踪分析，并在40微米的顺道追踪分析。 </li></ol>注意:FG标记的神经元可被观察为在紫外光下激发的白细胞。 BDA被O / N培养检测到具有抗生物素蛋白-生物素-过氧化物酶复合物和3,3&#39;二氨基联苯胺并加入镍中的0.4％，以提高纤维6,7的对比度。

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多年来，像缺血性脑卒中或脑外伤神经退行性疾病的研究主要集中在神经保护疗法，旨在促进急性中风期神经元存活的发展。当翻译到诊所绝大多数药物治疗已发现可有效地失败啮齿动物模型。这种情况的原因治疗失败包括但不限于缺乏导致持续功能性神经恢复持续药物作用。它制定战略促进大脑重塑，从长远来看是重要的。因为促进神经元存活的仅此还不足以让成功的中风恢复期，所建议的大量不成功的神经保护试验中，神经元可塑性的刺激，最近获得在该领域的主要兴趣。 用于药物输送的腹腔注射，尾部血管内我njection，股骨注射，单立体定向注射载体的进入脑，并继续恒定交付miniosmotic泵。后者可包括全身递送，如果泵不具有插管，或可以器官定向，如我们已经表明用于输送到大脑。除miniosmotic泵和使用病毒载体的，所有其他策略会诱发波动的药物浓度。对于长期实验中从而成为必要的动物提交于接收频繁注射的应力。血脑屏障强加的蛋白质或药物从血液中脑吸收的一个重要障碍，造成巨大的蛋白质或药物的剂量，以达到治疗浓度在大脑中的需要。例如佩莱格里尼等人在为30克（750 IU /天为300克的大鼠）的动物相当于75 IU /天的剂量递送的rhEPO通过腹膜内注射（2013）5。相比较而言，靶向递送rhEp的Ø大脑使我们能够使用的只有10 IU /天低得多的剂量在我们的研究成功中风恢复期，这使我们能够实现复苏在大的时间尺度为0.25微升/小时的固定利率。 在这项工作中，我们已经表明微泵植入的与连接到颅骨以便直接递送可塑性促红细胞生成素蛋白进入心室，从而绕过血脑屏障的套管的方法。通过这种方法，生成素促进神经恢复在许多方面，包括减少梗塞面积，减少神经胶质疤痕形成和诱导血管生成。促红细胞生成素也促进神经元存活并增加从对侧运动皮层的预测对失神经支配红核和面神经核。纤维的发芽揭示通过注射顺行道示踪剂BDA到运动皮层（ 图4A和5A）的。的功能关联到纤维的发芽是公关由运动技能的提高（ 图5B）ovided。此外，我们已经表明，可以应用于道示踪剂注入相同的方法来揭开丘脑-皮质连接通过注射的逆行示踪剂的FG（图6B）的。 在miniosmotic泵的制备中，关键是要考虑目标点与使用间隔物。我们使用一个间隔件由0.5mm至降低针的长度以这种方式在针的最前端是在给定的坐标与心室接触（-0.2毫米尾鳍，0.9毫米的横向，2.5毫米dorso腹侧，与对于前囟门）。然而，如果更深结构是研究的目标，则没有间隔物是必要的。同样地，如果一个以上的外部传递点所需的（ 即 ，皮质），则更多的间隔盘将是必要的。导管必须足够长，使泵是不是太贴近头部，因为它会阻碍谅解备忘录的运动本身，也不能太长，因为一旦植入过度长度可能会导致导管弯曲，从而通过鼠标的自然运动增加套管除去的危险。的2厘米导管的部分提供在流动性和植入物的稳定性（图1和2）而言非常好的结果。泵在37℃CO / N的培养允许泵立即开始泵送药物进入脑在植入的那一刻。 在miniosmotic泵植入关键是要保证该颅骨植入插管之前适当干燥。通常用70％乙醇的清洁会导致骨骼变干，但如果连续出血发现，有cauterizer轻轻抚摸头骨将彻底擦干。重要的是要保证引入针的是作为垂直和慢尽可能是至关重要的。一旦到位，虽然胶水是干燥，把手指上的套管顶部防止其侧向移动OV呃头骨。应特别注意给予套管的伤口和位置。重要的是，是不完全在颅骨的中间线，但稍微向右侧进行的切口。当闭合伤口，如果切口，在中间线，皮肤会超负荷，由此增加伤口开口的危险。稍微使切口一侧将允许缝合点从套管中最高的部分是离开。其结果是会有的缝合点不到紧张和伤口愈合正常。动物应单独笼并检查每天，尤其是在植入后的第10-15天。万一伤口裂开，伤口必须尽快封闭。如果套管是去除或动物呈现感染，实验已被终止。不建议重新植入插管。为成功植入用的组织广告足够量是非常重要的hesive（不要太多！），因为它降低了骨和增加的套管除去的危险。但是使用过少的粘合剂也将不会举行附着在骨套管。的miniosmotic泵可携带药物溶解在各种各样的物质，是唯一局限于此，所述溶剂是生物相容的。此外，由于体积小（200微升）必须确定实验所需的浓度是否合适，不会造成泵内沉淀。 道有两种顺行或逆行示踪剂追踪是一个非常完善的技术来研究大脑连通性和可塑性。注射，以确保目标的大脑区域中的一个希望学习精度时，必须小心考虑使用立体定位框架（注射皮质时即预防注射的胼胝体）。 对于所有手术干预和以减轻疼痛和炎，动物应该用0.1毫克/千克丁丙诺啡/公斤，每天一次，干预三天后治疗的干预和Caprofen之前在4毫克。 总之，本方法提供了用于研究的蛋白质或药理学化合物作用于脑损伤，即代表非常适用于对脑可塑性研究的方法的适当的工具。

The delivery of proteins and pharmacological compounds into the brain are important strategies for studying mechanisms underlying brain diseases and evaluating candidate molecules for new treatments 1,2. In experimental neurosciences, the delivery of vectors such as plasmids or adenoviruses has become an important tool for studying long-term actions of proteins in the brain 3,4. Single injections of vectors present the advantage of a system which by itself will maintain highly stable levels of the therapeutic agent in the brain 4. However, for long term experiments with purified drugs systemic administration by intraperitoneal injection induces stress in mice or rats, and is not the best choice when a targeted brain response is needed, requiring also large doses of drug5. Miniosmotic pumps represent an ideal system for prolonged direct drug delivery into the brain by circumventing both low accessibility to the brain and also peaks of drug concentration, as the delivery of the drug happens directly into a targeted place in the brain and at a fixed flow rate determined by the pump model that is chosen2,6,7. Indeed, this system has allowed us to successfully study brain recovery after stroke by delivery of several drugs such as recombinant human erythropoietin (rhEpo) and vascular endothelial growth factor 6,7. 
Brain plasticity is essential for the rewiring of connections in response to brain injuries. Plasticity is a broad concept that ranges from the formation or elimination of synaptic contacts, growth of dendritic spines and also elongation or retraction of long distance connections8,9. The brain was previously believed to not be capable of reconstructing connections after a lesion. However many approaches have shown that if properly stimulated it can reestablish connectivity 6,7,10. One technique that is particularly useful to study this is the use of tract tracers. Anterograde tract tracers are compounds that can enter neurons at the soma and then distribute all along the axons until these reach their target structures. Two examples are cascade blue (CB) and biotinylated dextran amine (BDA). Conversely, retrograde tract tracers, such as cholera toxin B (CTB) or fluorogold (FG) enter the neuron through the axon terminal and then distribute back to the soma thus revealing the site of origin of neurons targeting the injection site.
Here, we present the methods that we use for implantation of miniosmotic pumps for direct delivery of proteins or drugs that have potential effects on neural plasticity as well as the injection of BDA and FG to unveil input and output connections to the motor cortex. BDA will also be used as an example of a tract tracer used to demonstrate increased plasticity of axons emerging from the co after stroke under rhEpo treatment.

<table><tbody><tr><td>Alzet miniosmotic pump. Model 2004.</td><td>Alzet</td><td>000298</td><td>Drug container</td></tr><tr><td>Brain infusion kit 3 1-3 mm</td><td>Alzet</td><td>0008851</td><td>Drug brain delivery system</td></tr><tr><td>Loctite&amp;nbsp; 454 Prism gel&amp;nbsp;</td><td>Loctite</td><td>45404</td><td>Cyanoacrylate adhesive for cannula adhesion to the skull</td></tr><tr><td>75N glass syringe&amp;nbsp;</td><td>Hamilton</td><td>87900/00</td><td>Injection of tract tracers</td></tr><tr><td>Biotin Dextran Amine (10,000 MW)</td><td>Molecular probes</td><td>N-7167</td><td>Anterograde tract tracer</td></tr><tr><td>Fluorogold</td><td>Fluorochrome, LLC.</td><td></td><td>Retrograde tract tracer</td></tr><tr><td>Quintessential Stereotaxic Injector (QSI)</td><td>Stoelting</td><td>53311</td><td>Stereotactic device for coordinate determination, pump implantation and tract tracer injection.</td></tr></tbody></table>

implantation of miniosmotic pumps and delivery of tract tracers to study brain reorganization in pathophysiological conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

我们在30天内进行付运开始后3天提交的动物大脑中动脉闭塞30分钟由腔内缝合方法诱导在左纹状体的病变，然后输送的rhEPO直接进入脑由miniosmotic泵（图1， 图3）的装置中风6。 图4显示这是CB和BDA注射并且其中示踪剂注射后区跟踪的皮质脊髓束的示意图。我们发现后分别为14和42天内进行付运的rhEPO递送的握力和电机性能（图5）的改进。递送BDA成接收一个行程的左纹状体动物的右马达皮层，显示增加运动纤维交叉处的红色和面神经核的水平中间线（图5），显示出发芽纤维作为成功染色与miniosmot药物治疗的结果IC泵。促红细胞生成素治疗也增加了神经元的存活，延迟弥漫性星形胶质细胞，减少神经胶质瘢痕形成，并在学习期间6增加血管生成。通过使用此相同的技术对道示踪剂注入我们能够成功地检测在通过注射的逆行示踪剂的FG（图6），连接到皮质丘脑核。 <img alt="图1" src="/files/ftp_upload/52932/52932fig1.jpg" /> 图1.组件在本协议中使用的miniosmotic泵，间隔盘，套管和可移动的标签，导管，流量主持人和miniosmotic泵可以观察到。完全组装的泵的方面可以在图2A中可以看出。 <a href="https://www.jove.com/files/ftp_upload/52932/52932fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ithin页="1"&gt; <img alt="图2" src="/files/ftp_upload/52932/52932fig2.jpg" /> 图泵植入要点2.摘要。 （A）鼠标显示为放置在立体定向设备上，与旁边的完全构造miniosmotic泵。箭头表示选择用于植入的坐标。（B）中的泵已经介绍了关于动物背部和仅插管保持在外部。颅骨已经钻出。（℃）纵横植入后的头部。（C.1）的套管上的位置，但在可移动的标签并没有被切断。（C.2）的可移除的拉已被切割并缝合的伤口现在可以开始。（D）中的星号示出了最近植入动物相比，在30天之后注入（＃）的动物。当正确地安置，伤口应保持关闭状态，直到程序结束如所示的图像上。ove.com/files/ftp_upload/52932/52932fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52932/52932fig3.jpg" /> 图3. Nissl染色指示植入上皮层的部位，一个小切口可以在左侧皮层（箭头）的一部分来观察。穿透的区域的宽度是大约50微米。的是基于Nissl染色没有明显严重的组织改变相比于相应的对侧区域（*）。 R:右半球。 L:左脑半球。比例尺= 300微米。 <a href="https://www.jove.com/files/ftp_upload/52932/52932fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52932/52932fig4.jpg" /> 图4.道像以前一样通过Reitmeier 等 6,7公布的示踪剂注入的策略。 （一）示意图显示注射部位的道示踪剂BDA在对侧运动皮层，而CB注射到梗死半球的运动皮质。纤维接着向红核（未示出）和面神经核（ 参见图5）。（B）中的顺行道示踪剂BDA所述的注射部位旁的运动皮层被示出。注意红色箭头指示针道，而黑色箭头显示标记澳门汇业银行的几个皮层细胞。 CX:皮质。 CC:胼胝体。五:心室。 FN:面神经核。 B中比例尺= 200微米。 图4A转载使用一种许可6。 <a href="https://www.jove.com/files/ftp_upload/52932/52932fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <i毫克ALT ="图5"SRC ="/文件/ ftp_upload / 52932 / 52932fig5.jpg"/&gt; 在皮质延髓的纤维在面神经核的水平（前囟-5.8毫米至-6.3毫米）然后检测图5.恢复递送促红细胞生成素后的梗塞脑。（A） 的 BDA在对侧运动皮层注入。每个半球交线被画平行于中线和纤维交叉的方向上的各线到​​ipsilesional和对侧半球进行计数，并表示为总标纤维的皮质脊髓束的百分比。促红细胞生成素增加纤维口岸方向的对侧面神经核数据是手段+ - SD。数据用单向方差分析，随后通过至少显著差异测试，§P&lt;0.05与载体处理非缺血小鼠。（B）的电机行为显示的手的握力和协调在轮值杆测试的改善。数据是平均值VALUES + - SD。数据通过双向重复测量ANOVA分析，随后单向ANOVA /至少显著差异为每个时间点测试。 §P&lt;0.05与缺血前比较基准; * P &lt;0.05与车辆治疗缺血性小鼠。 图5A和5B转载使用一种许可6。 <a href="https://www.jove.com/files/ftp_upload/52932/52932fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52932/52932fig6.jpg" /> 注射荧光金（FG）的后到达图6的目标结构。（A） 中的FG旁边的运动皮层，如图1所示注射部位。注意红色箭头指示针轨道和白色箭头指示在几个标记的细胞皮层（B）中的FG注入电机附近皮质中对VPL检测。网络连接:伞。 IC:内囊。 RT:丘脑网状核。 VPL:丘脑腹后外侧核。比例尺= 200微米。 <a href="https://www.jove.com/files/ftp_upload/52932/52932fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions at JoVE.com

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Miniosmotic植入泵和交货道示踪研究在病理情况下脑重组

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

implantation-miniosmotic-pumps-delivery-tract-tracers-to-study-brain

Research

JoVE Journal

Neuroscience

14.2K 次观看.  University of Duisburg-Essen. In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

视频: Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

科研

生物学

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, ...

神经科学

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

医学

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

通过对流增强的传递将抗体输送到毛里脑

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and autism. As evidence suggests that remyelination can effectively ameliorate the disease symptoms, there is an increasing focus on drug development to promote the myelin regeneration process. Thus, a region-selectable and result-reliable drug delivery technique is required to test the efficiency and specificity of these drugs in vivo. This protocol introduces the osmotic pump implant as a new drug delivery approach in the lysolecithin-induced demyelination mouse model. The osmotic pump is a small implantable device that can bypass the blood-brain barrier (BBB) and deliver drugs steadily and directly to specific areas of the mouse brain. It can also effectively improve the bioavailability of drugs such as peptides and proteins with a short half-life. Therefore, this method is of great value to the field of central nervous system myelin regeneration research.

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.

基于渗透泵的药物递送用于中枢神经系统 体内 髓鞘再生研究

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and ...

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits is causal remains elusive. Moreover, whether these deficits are mediated by an increase in glucose levels in cerebrospinal fluid (CSF) and/or blood is also unclear. There are very few studies investigating the direct effects of high CSF glucose levels on central nervous system (CNS) function, especially on learning and memory, since current diabetes models are not sufficiently developed to address such research questions. This article describes a method to chronically increase CSF glucose levels for 4 weeks by continuously infusing glucose into the lateral ventricle using osmotic minipumps in mice. The protocol was validated by measuring glucose levels in CSF. This protocol increased CSF glucose levels to ~328 mg/dL after infusion of a 50% glucose solution at a 0.25 &#181;L/h flow rate, compared to a CSF glucose concentration of ~56 mg/dL in mice that received artificial cerebrospinal fluid (aCSF). Furthermore, this protocol did not affect blood glucose levels. Therefore, this method can be used to determine the direct effects of high CSF glucose on brain function or a specific neural pathway independently of changes in blood glucose levels. Overall, the approach described here will facilitate the development of animal models for testing the role of high CSF glucose in mediating features of Alzheimer&#39;s disease and/or other neurodegenerative disorders associated with diabetes.

This article describes a detailed protocol to increase glucose concentration in the cerebrospinal fluid (CSF) of mice. This approach can be useful for studying the effects of high CSF glucose on neurodegeneration, cognition, and peripheral glucose metabolism in mice.

渗透压微型泵植入增加小鼠脑脊液中葡萄糖浓度

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits ...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Source: Ajoy, R., Chou, S. Y. <a href="https://www.jove.com/v/56410">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the assembly of a micro-osmotic pump brain infusion system followed by its surgical implantation in the mouse skull. This system enables continuous delivery of potential drugs and facilitates the study of their effects on the brain and brain tissue.

小鼠模型中的连续药物输注系统:在小鼠大脑中植入微渗透泵输注系统以实现连续药物输送的外科手术

Source: Ajoy, R., Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the ...

Miniosmotic植入泵和交货道示踪研究在病理情况下脑重组

摘要

探索更多视频

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

在中央小鼠中枢神经系统的干细胞移植的多模态成像

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

脑植入的微型器件及周围组织的完整的组织学特性研究

方法以使对新生儿鼠害的腹头骨开颅手术

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

通过对流增强的传递将抗体输送到毛里脑

基于渗透泵的药物递送用于中枢神经系统体内髓鞘再生研究

渗透压微型泵植入增加小鼠脑脊液中葡萄糖浓度

小鼠模型中的连续药物输注系统:在小鼠大脑中植入微渗透泵输注系统以实现连续药物输送的外科手术

Miniosmotic植入泵和交货道示踪研究在病理情况下脑重组

摘要

探索更多视频

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

在中央小鼠中枢神经系统的干细胞移植的多模态成像

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

脑植入的微型器件及周围组织的完整的组织学特性研究

方法以使对新生儿鼠害的腹头骨开颅手术

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

通过对流增强的传递将抗体输送到毛里脑

基于渗透泵的药物递送用于中枢神经系统 体内 髓鞘再生研究

渗透压微型泵植入增加小鼠脑脊液中葡萄糖浓度

小鼠模型中的连续药物输注系统:在小鼠大脑中植入微渗透泵输注系统以实现连续药物输送的外科手术

基于渗透泵的药物递送用于中枢神经系统体内髓鞘再生研究