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.
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.
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.
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 authors have nothing to disclose.
This work was supported by the Dr. Werner Jackstädt 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).
Alzet miniosmotic pump. Model 2004. | Alzet | 000298 | Drug container |
Brain infusion kit 3 1-3mm | Alzet | 0008851 | Drug brain delivery system |
Loctite 454 Prism gel | Loctite | 45404 | Cyanoacrylate adhesive for cannula adhesion to the skull |
75N glass syringe | Hamilton | 87900/00 | Injection of tract tracers |
Biotin Dextran Amine (10000 MW) | Molecular probes | N-7167 | Anterograde tract tracer |
Fluorogold | Fluorochrome, LLC. | Retrograde tract tracer | |
Quintessential Stereotaxic Injector (QSI) | Stoelting | 53311 | Stereotactic device for coordinate determination, pump implantation and tract tracer injection. |