通过对流增强的传递将抗体输送到毛里脑
Summary
对流增强输送(CED)是一种通过直接灌注大量组织,使治疗药物有效输送到大脑的方法。该过程需要使用导管和优化的注射程序。该协议描述了一种将抗体送入小鼠大脑的CED方法。
Abstract
对流增强输送 (CED) 是一种神经外科技术,利用导管系统能够有效灌注大脑容量。这种方法提供了一种安全分娩方法,通过血脑屏障(BBB),从而允许治疗与不良BBB渗透性或那些不需要全身接触,例如,由于毒性的治疗。CED 要求优化导管设计、注射协议和内福的特性。通过该协议,我们描述了如何执行含有高达20μg的抗体的溶液的CED到小鼠的牛精。它描述了步进导管的制备,在体外测试,并使用斜坡注射程序在小鼠中执行CED。该协议可以很容易地调整其他输注量,并可用于注射各种示踪剂或药理活性或非活性物质,包括化疗剂、细胞因子、病毒颗粒和脂质体。
Introduction
血脑屏障 (BBB) 形成一个半渗透边界,将中枢神经系统 (CNS) 与血液循环分离。然而,在各种疾病的背景下,如脑肿瘤、阿尔茨海默氏病(AD)或帕金森病(PD)等疾病,达到CNS是必要的。这成为新疗法开发的重要,特别是如果被测试的药物表现出较差的BBB渗透性或其全身暴露可能导致危险的毒性1,2。一些临床使用的抗体显示这两个功能。解决这个问题的方法是直接在BBB后面提供治疗药物。
对流增强输送(CED)是一种神经外科技术,能够有效灌注大脑容量。这是通过在目标区域通过手术安装一个或多个导管来实现的。在药物应用过程中,导管开口处形成压力梯度,成为组织3、4中迷剂的驱动力。因此,决定灌注范围2、4、5的不是输液的持续时间,而不是扩散系数。与传统的基于扩散的脑内注射方法2、6相比,这提供了在更大的脑体积上均匀地输送的内福剂。同时,这种分娩方式具有较低的组织损伤风险2。因此,CED 可以安全有效地给常规化疗药物用于治疗中枢神经系统肿瘤,以及提供免疫调节剂或许多其他中枢神经系统疾病中的抗性和对抗性抗体2 ,7,8,9.CED目前正在测试帕金森病,阿尔茨海默氏病,以及高档胶质瘤2,7,8,10,11的疗法。
导管设计和注射方案是影响CED 10、12、13、14、15、16结果的最重要因素之一。此外,它要求特定的物理化学性质,包括中等大小的颗粒,一个电子电荷,和低组织亲和力10,17。 这些参数中的每一个都必须根据大脑区域的组织特征进行潜在的调整,以瞄准2,10,17。
在这里,我们描述了将抗体溶液的CED执行到小鼠的牛锥(纹状体)的方法。此外,该协议包括在实验室设置中制备阶梯导管,在体外测试和执行CED。
文献中有多种导管设计,不同形状的导管、所用材料和导管开口数 12、15、18、19、20 ,21,22.我们使用的阶梯导管由熔融的二氧化硅毛细管制成,从钝端金属针伸出1毫米。这种导管设计可以很容易地在研究实验室制造,并可重复提供良好的CED结果,当在体外测试与基因胶块与物理参数类似于脑气囊在体内23。
此外,我们实施斜坡方案,在体内提供5μL的芬福。在这样的协议中,注射率从0.2μL/min提高到最大0.8μL/min,从而最大限度地减少了导管内液倒流的机会以及组织损伤的风险16。使用该协议,我们在11分钟30秒内成功地在5μL的PBS中用高达20μg的抗体给小鼠。
该协议可以很容易地调整其他输注量或注射各种其他物质,如化疗,细胞因子,病毒颗粒或脂质体2,10,14,18 ,22.如果使用与磷酸盐缓冲盐水 (PBS) 或人工脑脊液 (aCSF) 抗体溶液相比具有巨大物理化学特性的富化液,建议采取额外的验证步骤。对于导管组件、验证和 CED,我们使用一个立体定向机器人描述所有步骤,该机器人的钻头和喷射单元安装在常规立体定向框架上。此过程也可以通过连接到可编程微输液泵的手动立体定向框架执行,该泵可驱动所述玻璃微注射器。
Protocol
此处描述的所有方法均已获得瑞士州兽医局批准,许可证号为 ZH246/15。 1. 步进导管的准备 为导管步骤制备熔融石英管 将内径为 0.1 mm、壁厚为 0.0325 mm 的熔融硅毛细管切割至 30 mm 的长度。 使用微锻件检查管道有无裂纹和热抛光,以确保管开口表面光滑。 内管固定在金属针中 将 27 G 针头安装在 10 μL 注射器…
Representative Results
该协议允许制备步进导管 (图 1),用于实验室环境中的 CED 程序。为了控制导管的泄漏、沿着针道回流和堵塞,我们建议将染料(例如锥形蓝色溶液)注射到腺苷酸块中。图 3描述了使用 CED 导管在 0.5 μL/分钟下注入 1 μL 后形成锥形蓝色云(图 3A)。在导管步骤的开头,看不到沿着针道的回流。此外,分散的云形成了一个所需的球形。这与使?…
Discussion
对流增强的输送,或压力介导的药物输注到大脑,首次提出在1990年初3。这种方法有望以可控的方式灌注血脑屏障后面的大脑容量2。然而,到目前为止,只有少数临床试验已经使用这种方法,部分是因为CED在临床设置已被证明是技术要求24,25。导管设计和输液方案的最新发展似乎已经克服了这些技术困难8,19。</s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了苏黎世大学(FK-15-057)、诺华医学生物学研究基金会(16C231)和瑞士癌症研究(KFS-3852-02-2016,KFS-4146-02-2017)对约翰内斯·沃姆·伯格和BRIDGE概念证明(20B1-1)的资助。#177300) 给琳达·舍尔哈默。
Materials
| 10 μL syringe | Hamilton | 7635-01 | |
| 27 G blunt end needle | Hamilton | 7762-01 | |
| Agarose | Promega | V3121 | |
| Atipamezol | Janssen | ||
| Bone wax | Braun | 1029754 | |
| Buprenorphine | Indivior Schweiz AG | ||
| Carprofen | Pfizer AG | ||
| Dental drill bits, steel, size ISO 009 | Hager & Meisinger | 1RF009 | |
| Ethanol 100% | Reuss-Chemie AG | 179-VL03K-/1 | |
| Fentanyl | Helvepharm AG | ||
| FITC-Dextran, 2000 kDa | Sigma Aldrich | FD2000S | |
| Flumazenil | Labatec Pharma AG | ||
| Formaldehyde | Sigma Aldrich | F8775-500ML | |
| High viscosity cyanoacrylate glue | Migros | ||
| Iodine solution | Mundipharma | ||
| Medetomidin | Orion Pharma AG | ||
| Microforge | Narishige | MF-900 | |
| Midazolam | Roche Pharma AG | ||
| Ophthalmic ointment | Bausch + Lomb | Vitamin A Blache | |
| PBS | ThermoFischer Scientific | 10010023 | |
| Polyclonal goat anti-rat IgG (H+L) antibody coupled with Alexa Fluor 647 | Jackson Immuno | ||
| Scalpels | Braun | BB518 | |
| Silica tubing internal diameter 0.1 mm, wall thickness of 0.0325 mm | Postnova | Z-FSS-100165 | |
| Stereotactic frame for mice | Stoelting | 51615 | |
| Stereotactic robot | Neurostar | Drill and Injection Robot | |
| Succrose | Sigma Aldrich | S0389-500G | |
| Topical tissue adhesive | Zoetis | GLUture | |
| Trypan blue | ThermoFischer Scientific | 15250061 | |
| Water | Bichsel | 1000004 |
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Cite This Article
Beffinger, M., Schellhammer, L., Pantelyushin, S., vom Berg, J. Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery. J. Vis. Exp. (149), e59675, doi:10.3791/59675 (2019).