Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Gerhard Leinenga; Liviu-Gabriel Bodea; Wee  Kiat Koh; Rebecca  M. Nisbet; J&#252;rgen G&#246;tz

doi:10.3791/61372

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

使用聚焦扫描超声将抗体输送到大脑中

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07:34 min

July 18, 2020

DOI: 10.3791/61372-v

Gerhard Leinenga¹, Liviu-Gabriel Bodea¹, Wee Kiat Koh¹, Rebecca M. Nisbet¹, Jürgen Götz¹

¹Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute,The University of Queensland

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a method for transiently opening the blood-brain barrier (BBB) in mouse models to facilitate the delivery of fluorescently-labeled antibodies and to activate microglia. The method allows for noninvasive delivery and monitoring of antibody uptake in the brain through histological techniques.

Key Study Components

Area of Science

Neuroscience
Immunology
Therapeutic delivery

Background

The blood-brain barrier limits the uptake of therapeutic antibodies to the brain.
Microglia play a critical role in brain immune responses.
Current methodologies for BBB opening and antibody delivery are limited.
Fluorescent labeling aids visualization of antibody uptake and microglia activation.

Purpose of Study

To develop a protocol to transiently open the BBB for antibody delivery.
To activate microglia in a controlled experimental setting.
To evaluate the effectiveness of this delivery method through histology.

Methods Used

The methodology involves focused ultrasound and microbubble injection into the mouse brain.
The main biological model utilized is anesthetized mice.
Key steps include the preparation of microbubbles and antibodies, and careful ultrasound targeting.
Fluorescent antibodies are purified and visualized to measure interaction with microglial cells.

Main Results

The technique successfully demonstrated antibody delivery to the brain.
Microglia exhibited increased phagocytic activity following antibody administration.
Visualization techniques confirmed effective localization and uptake of antibodies.
Scanning patterns enabled targeting of specific brain regions, broadening potential therapeutic applications.

Conclusions

This study provides a reliable method for enhancing therapeutic antibody delivery across the BBB.
It opens avenues for future research in drug development and understanding brain mechanisms.
The findings highlight the potential for this method in studying neuroimmunological interactions.

Frequently Asked Questions

What are the advantages of this antibody delivery method?

The method allows for noninvasive delivery of antibodies into the brain, which enhances therapeutic potential while minimizing surgical risks associated with traditional methods.

How is the biological model implemented in this study?

Anesthetized mice are used as the biological model, allowing researchers to conduct precise interventions without distress to the animals.

What types of data are obtained using this method?

The study provides data on the concentration and localization of antibody uptake in the brain, alongside changes in microglial activity indicated by histological staining.

How can this method be adapted for other studies?

This technique can be adjusted to target different brain regions or to investigate other therapeutic agents, thus broadening its application in neuroscience research.

Are there limitations to this approach?

While effective, the method requires careful control of ultrasound parameters to avoid potential damage to surrounding brain tissue and must be used with caution in studies.

这里介绍的是一种方案，用于局部或整个小鼠大脑短暂打开血脑屏障（BBB），以传递荧光标记的抗体并激活小胶质细胞。还提出了一种通过组织学检测抗体递送和小胶质细胞活化的方法。

只有一小部分治疗性抗体被大脑吸收。使用我们的方法，抗体通过瞬时打开血脑屏障输送到大脑中。这项技术使我们能够以无创方式将抗体输送到小鼠大脑中。

要使用细胞计数器对微气泡进行质量控制，请从汞齐调器中取出微气泡溶液，并使用 19 号针刺穿微气泡溶液样品瓶的隔膜。使用配备有 19 号针头的 1 毫升注射器在 5 毫升过滤流液中稀释 100 微升微泡，并将 100 微升稀释的微泡溶液吸取到比色皿中的 10 毫升过滤流液中。将比色皿锁定在细胞计数仪平台上，并确保使用 30 微米的孔径进行样品采集。

在软件中，加载标准作方法，然后选择编辑标准作方法和浓度。输入 5， 000 倍的稀释度，然后单击应用和确定。单击 edit info 以选择合适的文件名。选择预览并验证微泡样品浓度是否小于 10%选择 Start（开始）和 OK（确定）开始样品采集。

测量后，用过滤的流液冲洗细胞计数仪的孔径。在水浴中对稀释的微泡溶液的比色皿进行超声处理 30 秒。如图所示测量超声处理的微气泡溶液，然后单击 Edit info（编辑信息）将数据标记为空白。

测量空白后，从初始读数中减去最终读数，以排除任何不是微气泡的颗粒。要使用荧光抗小鼠 IgG 抗体标记切片，请按照制造商的说明，用 Alexa Flour 647 在 0.1 摩尔碳酸氢钠缓冲液中标记 1 毫克小鼠 IgG 抗体，在室温下放置 15 分钟。孵育结束时，将荧光标记的抗体溶液加载到离心柱上，并通过离心纯化抗体。

然后使用分光光度计测量蛋白质浓度。要设置聚焦扫描超声系统，请在水丸上增加 5 毫米的空间，将超声焦点放置在水丸底部下方 9 毫米处。用大约 300 毫升脱气去离子水填充水团，然后将环形阵列放入装满的水团中。

使用牙科镜检查表面是否有气泡，然后启动应用软件。在波形菜单中，选择设置波形占空比，并将脉冲重复频率设置为 10 赫兹，占空比为 10%，焦点为 80 毫米，中心频率为 1 兆赫兹，振幅为 0.65 兆帕，机械指数为 0.65。按 set 定义波形并将波形存储在内存中。

选择治疗计划后，打开运动控制器窗口中的扫描选项卡，输入 X 维度运动的开始、停止和增量值，以及 Y 方向运动的开始、停止和增量值。要定义治疗部位的作，请单击 event 并打开脚本编辑窗口。选择将按在每个治疗地点选择的顺序执行的作列表，并将移动类型设置为栅格网格。

在 events 选项卡中，选择 add actions 并将 move syncly start trigger ARB wait 和 stop trigger ARB actions 移动到脚本面板。然后单击 wait action 并选择 6， 000 毫秒的等待时间。为了准备动物进行分析，在确认麻醉小鼠对脚趾捏没有反应后，使用永久性记号笔标记头部中心，然后将保鲜膜粘在底部截止的小称重船的底部，并用超声凝胶填充称重船。

对于聚焦超声治疗，倒置小瓶微气泡，并将每克小鼠体重的 1 微升溶液轻轻加载到 29 号胰岛素注射器中。向注射器中加入 100 μL 荧光标记抗体，然后用拇指和食指轻轻倒置和滚动注射器，以混合抗体和注射器内的微气泡。小心缓慢地将 150 μL 微气泡和抗体溶液逆眶注射到小鼠体内，并设置计时器 2 分钟。

涂抹眼药膏，然后将鼠标放入头架中，并将其鼻子固定在支架上。将装满超声凝胶的小称量船放在头顶上，降低水球，直到它位于称量船内的超声凝胶顶部。然后使用纵杆在头部中心内直观地对准传感器焦点。

当计时器关闭时，在软件的运动选项卡中，选择重置原点并选择完整扫描。治疗完成后，将眼药膏涂抹在动物的眼睛上，并将小鼠置于温暖的恢复室中。这里显示了正确产生微气泡时可以获得的尺寸和浓度的培养计数器测量的代表性结果。

使用红外扫描仪或切片的荧光显微镜，可以很容易地在整个大脑或组织切片中观察大脑对抗体的摄取。如这些图像所示，小胶质细胞标志物的代表性染色可用于确定小胶质细胞在抗体递送后是否变得更加吞噬。确保动物的福利和安全非常重要，尤其是在进行眼眶后注射和将换能器应用于小鼠头部上方时。

这种方法允许应用各种扫描模式来针对大脑的不同区域，例如海马体、皮层或单个大脑半球。该技术可用于开发穿透血脑屏障的新疗法和药物，并研究血脑屏障形成和打开的机制。

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