我们感谢Clem Jones AO博士的遗产，澳大利亚国家卫生和医学研究委员会[GNT1145580，GNT1176326]，金属基金会和昆士兰州政府（DSITI，科学，信息技术和创新部）的支持。

所有动物实验均获得昆士兰大学动物伦理委员会的批准。
1. 内部微气泡制备
<ol><li>称出1，2-二硬脂酰-sn-甘油-3-磷酸胆碱和1，2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-[氨基（聚乙二醇）-2000]（铵盐）的摩尔比为9:1。每1mL微泡溶液需要0.5mg脂质混合物。或者，脂质已经以氯仿购买，如果使用预溶解的脂质，则继续进行步骤1.3。</li>
	<li>将脂质溶解在玻璃烧杯中的少量氯仿中。</li>
	<li>用蒸发器或氮气流蒸发氯仿。</li>
	<li>用10 mL PBS + 10%甘油+ 10%丙二醇溶液重新水合干燥的脂质膜，该溶液已通过0.22μm过滤器过滤。</li>
	<li>将再水化的脂质溶液置于设置为55°C（高于脂质的熔化温度）的水浴超声仪中，并超声处理直至完全溶解。</li>
	<li>等分脂质溶液放入高压灭菌的1.5 mL HPLC小瓶中，并拧在隔膜盖上。</li>
	<li>用装有27 G针头的5 mL注射器吸入小瓶中的所有空气，并在小瓶中产生真空。</li>
	<li>用随附的注射器将八氟丙烷加入小瓶中，从罐中吸取气体。通过读取注射器中的体积，用1-2mL八氟丙烷填充小瓶。</li>
	<li>用石蜡膜密封每个小瓶并冷藏。</li>
	<li>在实验当天，将小瓶置于室温，向小瓶中加入0.5mL的0.9%NaCl溶液，然后将小瓶放入混合器中并搅拌45秒（预设时间）以产生微气泡。</li>
</ol>2. 使用犁刀计数器进行微气泡质量控制
<ol><li>将微气泡溶液从汞合金中取出，并通过用19 G针刺穿隔膜来从小瓶中排出气体。</li>
	<li>通过执行两步1:5，000连续稀释来稀释微泡溶液，方法是使用1ml注射器和19 G针头将100μL微气泡溶液加入5mL过滤流动溶液中，然后取100μL1:50稀释微气泡溶液并使用移液管移液到比色皿中的10mL过滤流动溶液中。</li>
	<li>检查电解质罐是否有足够的流动溶液，并且废液罐是空的。</li>
	<li>将比色皿放在犁刀柜台平台上，并将其锁定到位。使用30 μm孔径进行样品采集。</li>
	<li>在软件中，加载标准操作方法 （SOM），然后选择 编辑 SOM |浓度。 输入5000x稀释液。</li>
	<li>在软件中，选择合适的文件名。例如，microbubble_1_date。</li>
	<li>将比色皿装载并固定到平台中。</li>
	<li>在软件中，选择" 运行|预览 并验证样品浓度是否小于 10%。如果该数字高于10%，则以更高的稀释因子对微气泡进行新的稀释。</li>
	<li>选择"开始"开始采集样品以获得初始读数。</li>
	<li>每次测量后，用过滤后的流动溶液冲洗库尔特计数器的孔径。重复步骤 2.2-2.9 以获得 3 个重复。</li>
	<li>将含有稀释微气泡溶液的比色皿放入超声器水浴中，超声处理30秒。</li>
	<li>用超声微气泡测量溶液，并标记为空白。</li>
	<li>在软件中，从初始读数中减去最终读数。这减去任何不是微气泡且不含气体的颗粒。</li>
	<li>在软件中选择 显示结果 ，以显示微气泡浓度、大小分布、平均大小和体积浓度。</li>
</ol>3. 荧光抗体标记
<ol><li>在PBS中获得1mg / mL的小鼠IgG溶液，不含任何添加剂。</li>
	<li>按照试剂盒中制造商的说明，在0.1 M碳酸氢钠缓冲液中用AlexaFluor 647标记1mg小鼠IgG。该量的荧光标记IgG足以在5-7只成年小鼠上执行该过程，其中施用5mg / kg剂量的抗体。</li>
	<li>将染料加入IgG溶液中0.1M碳酸氢钠缓冲液中，并在室温下孵育15分钟。</li>
	<li>通过将抗体溶液移液到离心柱中并以1，000 ×g 离心5分钟来纯化荧光标记的抗体。免费染料将保留在柱床中。</li>
	<li>使用分光光度计测量蛋白质浓度。测量共轭溶液在280nm和650nm（A280和A650）处的吸光度。使用以下公式计算样品中蛋白质的浓度: 蛋白质浓度 （M）= [A280 - （A650 x 0.03）] x 稀释因子 / 203，000。</li>
	<li>使用分光光度计计算使用以下公式的标记程度: 每摩尔蛋白质的摩尔染料 = A650 x 稀释因子 / 239 000 x 蛋白质浓度 （M）。 注意:可接受的标记度是每摩尔蛋白质3-7摩尔染料，通常获得的标记度约为6。</li>
</ol>4. 超声设置
<ol><li>使用聚焦超声系统，将5mm垫片加入水推注器中，将超声聚焦置于水推注底部下方9mm处。</li>
	<li>用约300 mL去离子水填充水，该去离子水已用在线脱气器脱气20分钟（氧气含量应低于3 ppm）。将环形阵列放入填充的水推注中，并使用牙科镜子检查表面上没有气泡。如果表面上存在，请取出环形阵列，并在水中将其更换。</li>
	<li>启动应用程序软件。在波形菜单中，选择 设置波形占空比。设置为PRF（Hz）10，占空比10%，聚焦80 mm，中心频率1 MHz，振幅（MPa峰值负压）0.65 MPa，机械指数= 0.65。按 Set 定义波形并将其存储在内存中。 注:聚焦超声系统由制造商根据经过校准的水听器进行的测量进行预校准。</li>
	<li>在聚焦超声系统软件中，定义治疗计划。这需要定义一个由多个单独的治疗部位组成的治疗区域，并确定在每个治疗地点要采取的行动。在这种情况下，治疗区是小鼠大脑的一个半球。</li>
	<li>在运动控制器窗口中，转到 "扫描 "选项卡， 然后输入 x 维度中运动的开始、停止和递增值，以及 y 方向运动的开始、停止和递增值。输入 X:开始 -4、停止 3.50 和 Y:-3.00、停止 3、增量 1.5、# 循环:1 的值。</li>
	<li>定义治疗部位的操作。在运动控制器窗口中，选择"事件"按钮。在"脚本编辑窗口"中，选择将按照在每个治疗部位选择的顺序执行的操作列表。将"移动类型"设置为脚本窗口顶部的栅格网格。在事件选项卡中，选择"添加动作"以将其移动到脚本面板，然后添加"同步移动"、"启动触发器 arb"、"等待"、"停止触发器 arb"。单击等待操作，然后选择 6， 000 毫秒的等待时间。 注意:这些设置将使治疗成为一个6 x 5的治疗点网格，间隔1.5毫米，每个点的治疗持续时间为6秒。对小鼠大脑进行超声处理的总持续时间约为3分钟。这种尺寸的治疗网格适用于体重约30克的成年C57 / Bl6小鼠。治疗点网格的大小可以根据鼠标的大小向上或向下调整。</li>
</ol>5. 动物准备
<ol><li>用精确到0.1g的天平称量鼠标。</li>
	<li>用 90 mg/kg 氯胺酮和 6 mg/kg 西拉嗪腹膜麻醉小鼠。用脚趾捏住测试无反射。或者，可以使用适当的吸入麻醉装置和适当的面罩用异氟醚麻醉小鼠。如果使用异氟醚，应在超声检查期间将小鼠放在加热垫上以防止体温过低。</li>
	<li>使用电动剃须刀剃掉动物头部的毛发，然后用棉签涂抹脱毛膏，停留2-3分钟或直到用湿纱布擦拭头发干净。注意脱毛霜不会进入老鼠的眼睛。</li>
	<li>用永久性标记标记鼠标头部的中心。换能器的中心有一个孔，换能器的焦点和焦点可以在视觉上对齐。</li>
	<li>将以前底部切断的小称重船装满，并用超声波凝胶粘在称重船底部的保鲜膜代替。这可以用作8 mm的垫片，并提供与鼠标头部的良好耦合，并允许目视检查与鼠标头部对齐的换能器的焦点。</li>
</ol>6. 微气泡制备
<ol><li>将微泡小瓶加热至室温。要激活，将0.5mL0.9%NaCl溶液加入小瓶中，并放入混合器中搅拌45秒以产生微气泡。</li>
	<li>通过用27 G针刺穿隔膜来排出小瓶。</li>
</ol>7. 超声治疗
<ol><li>倒置微气泡的小瓶，轻轻地吸取溶液的1μL / g体重。向该溶液中加入荧光标记的抗体并在注射器中轻轻混合。注射的最大体积为150μL。 注意:内部制备的微气泡的浓度比临床使用的微气泡低约60倍（例如，定义微气泡）。调整体积或浓度，使注射的微气泡数量与临床上使用的微泡相似（即。定义1.2 x 108 微气泡/ kg体重）。</li>
	<li>逆向轨道注射微气泡和抗体溶液，注意轻柔缓慢地注射。然后，将眼药膏涂抹在小鼠的眼睛上。</li>
	<li>将鼠标放在头架中（请参见材料表）并固定鼠标的鼻子。然后将充满超声波凝胶的小称重船放在头顶上。</li>
	<li>降低推注水量，直到它位于称重船中的超声凝胶顶部。</li>
	<li>使用操纵杆将换能器焦点移动到头部中心。在运动选项卡中选择 重置原点 。</li>
	<li>选择完成扫描。步骤 7.3-7.6 需要 2 分钟。</li>
	<li>为了保持一致性，请设置计时器以确保在注射微气泡和选择完整扫描之间有2分钟的延迟。</li>
	<li>治疗完成后，将眼药膏涂抹在眼睛上，并将小鼠置于温暖的恢复室中。如果在手术过程中观察到体温过低，可以在小鼠下方放置一个加热垫，以便在手术过程中提供补充热量。</li>
</ol>8. 组织采集和加工
<ol><li>在超声给药后的目标时间点（至少1小时以检测大脑中的高水平抗体），用过量的戊巴比妥溶液（100mg / kg）深度麻醉小鼠，并用30mL PBS经心电泳灌注小鼠。需要良好的灌注来特异性检测已递送到大脑的荧光标记抗体。</li>
	<li>收集大脑并通过浸泡在4%PFA中在4°C下固定24小时，然后用PBS洗涤。</li>
	<li>通过将大脑放在托盘上并在700nm通道中获取图像，在红外扫描仪中对大脑进行成像。 注意:固定后，将大脑从PFA中取出，在PBS中洗涤并切片。或者，可以将其置于乙二醇冷冻保护剂溶液中，在4°C下或直到浸没24小时，然后移动到含有小瓶的新型冷冻保护剂中并置于-20°C下长期储存。</li>
	<li>使用振动切片机在PBS中切除脑冷。将大脑粘在平台上，切割30-40μm切片并收集到PBS中。 注意:溶酶体自发荧光（在大约12个月以上的动物的切片中普遍存在）应通过在光室箱中，室温和含有叠氮化物（0.02%）的PBS中过夜照明来漂白切片，以阻止细菌生长。</li>
</ol>9. 组织染色和图像采集
<ol><li>在室温下将切片转移到封闭溶液（5%BSA，0.2%Triton / PBS）2小时，然后用0.2%Triton / PBS替换溶液，将切片洗涤3x。</li>
	<li>将切片在4°C下孵育过夜，加入针对Iba1（稀释1:1，000）和CD68（稀释1:500）的一抗，在0.2%Triton / PBS中，然后用0.2%Triton / PBS洗涤3次。</li>
	<li>用荧光二抗（稀释1:500）在室温下孵育切片2小时，然后仅用0.2%Triton / PBS洗涤3次。 注意:也可以使用DAPI溶液（0.5μg/ mL）染色细胞核。</li>
	<li>将截面转移到带有硬设置安装介质的幻灯片和盖玻片上。在可视化和图像采集（过夜）之前，留出足够的时间让安装介质固化。</li>
	<li>使用共聚焦显微镜和至少40倍放大倍率的物镜，获得超声靶向脑区域的z-stack图像（至少10μm深度和0.3μm步长，每只动物10张图像）。 注:小心在信号动态范围内采集图像，避免曝光不足/过度曝光。以显微镜使用的相应软件格式保存图像文件。</li>
</ol>10. 图像分析
<ol><li>使用图像分析软件文件导入程序转换 z 堆栈显微镜文件。</li>
	<li>将转换后的文件打开到软件中，并调整Iba1通道的强度以观察小胶质细胞。</li>
	<li>通过在单个小胶质细胞周围绘制一个框来裁剪单个小胶质细胞，选择"裁剪"并保存新文件。</li>
	<li>通过以下方式构建 IBA1 信号的 3D 表面渲染:<ol>
<li>打开在上一步中生成的单单元格 Imaris 文件。</li>
		<li>将曲面添加到文件中。</li>
		<li>选择与 Iba1 染色相对应的通道。</li>
		<li>应用应与 Iba1 染色重叠的阈值</li>
		<li>仅选择正在形成目标小胶质细胞的感兴趣结构</li>
	</ol>
</li>
	<li>完成流程<ol>
<li>获取并记录 Iba1 染色的体积</li>
		<li>构建 CD68 染色的 3D 表面渲染</li>
		<li>在 IBA1 表面子文件内，遮盖 CD68 通道并移除 IBA1 染色外部的体素</li>
		<li>向文件添加新曲面</li>
		<li>选择与CD68染色相对应的通道</li>
		<li>应用应与CD68染色重叠的阈值，并尽可能与染色体积相匹配</li>
		<li>完成该过程，因为只有小胶质细胞内CD68结构将存在于该文件中，因此，它们不需要另一个阈值过滤步骤。</li>
		<li>获取并记录CD68阳性结构的数量和平均体积</li>
		<li>根据相应的IBA1结构计算CD68结构的相对体积，作为吞噬状态下小胶质细胞活化的量度。</li>
	</ol>
</li>
</ol>

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<li>Götz, J., Halliday, G., Nisbet, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Pathogenesis+of+the+Tauopathies%5BTitle%5D)+AND+%22Annual+Reviews+of+Pathology%22%5BJournal%5D)">Molecular Pathogenesis of the Tauopathies.</a> Annual Reviews of Pathology. 14, 239-261 (2019).</li>
<li>Pandit, R., Leinenga, G., Götz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repeated+ultrasound+treatment+of+tau+transgenic+mice+clears+neuronal+tau+by+autophagy+and+improves+behavioral+functions%5BTitle%5D)+AND+%22Theranostics%22%5BJournal%5D)">Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.</a> Theranostics. 9 (13), 3754-3767 (2019).</li>
<li>Karakatsani, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unilateral+Focused+Ultrasound-Induced+Blood-Brain+Barrier+Opening+Reduces+Phosphorylated+Tau+from+The+rTg4510+Mouse+Model%5BTitle%5D)+AND+%22Theranostics%22%5BJournal%5D)">Unilateral Focused Ultrasound-Induced Blood-Brain Barrier Opening Reduces Phosphorylated Tau from The rTg4510 Mouse Model.</a> Theranostics. 9 (18), 5396-5411 (2019).</li>
<li>Valdez, M. A., Fernandez, E., Matsunaga, T., Erickson, R. P., Trouard, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution+and+Diffusion+of+Macromolecule+Delivery+to+the+Brain+via+Focused+Ultrasound+using+Magnetic+Resonance+and+Multispectral+Fluorescence+Imaging%5BTitle%5D)+AND+%22Ultrasound+in+Medicine+and+Biology%22%5BJournal%5D)">Distribution and Diffusion of Macromolecule Delivery to the Brain via Focused Ultrasound using Magnetic Resonance and Multispectral Fluorescence Imaging.</a> Ultrasound in Medicine and Biology. 46 (1), 122-136 (2020).</li>
</ol>

我们没有什么可披露的。

荧光标记的抗体可以使用聚焦超声和扫描模式下施加的微气泡一起传递到大脑。抗体递送、小胶质细胞形态和溶酶体肿大可通过超声扫描后的荧光显微镜检测。小胶质细胞可以吸收到其溶酶体抗体和抗原中，抗体在Fc受体介导的过程中结合4。
使用这种方法实现可重复的BBB开放和抗体递送有许多关键步骤。确保超声换能器和鼠标头部之间的良好耦合至关重要。去除鼠标头上的所有毛发，并确保接头中没有气泡。内部制备的微气泡的特性是成功的关键。微气泡必须具有108 微气泡/mL的足够浓度和尺寸分布，使得超过90%的微气泡直径低于10μm。这是因为已知较大的微气泡会被肺部从循环中过滤掉。可接受的中位数大小在1-3μm之间。微气泡必须轻轻处理和注射，以避免在注射器中破坏它们。重要的是，超声波在注射微气泡后不迟于2分钟，这可以通过实践来实现。使用SUS方法可以轻松实现整个大脑或整个半球的靶向，并且在靶向大区域时，靶向的准确性不太可能成为问题。较小的大脑区域（如海马体或纹状体）也可以成功靶向，但在这种情况下，焦点与目标区域重叠很重要。使用典型的超声换能器，小鼠大脑的高度与聚焦超声束在1 MHz处的轴向长度相似，因此换能器只需要在x和y维度上移动，而不是z维度。这可以通过了解特定大脑结构的立体定位坐标以及通过观察小鼠脱毛皮肤的lambdoid和矢状缝合线来确定。
在这里，我们展示了一种使用眶后注射来传递微气泡和抗体的技术。眶后注射的替代方法是尾静脉注射，这也是一种传递微气泡和抗体的有效技术。眶后注射的优点是在技术上比尾静脉注射更具挑战性，并且可以重复多次（交替注射眼），组织损伤的风险非常小。
如果在大脑中没有检测到荧光标记的抗体，则可能是BBB没有打开。故障排除应侧重于获得浓缩的微气泡溶液并注射，以免破坏微气泡，并在注射时间的两分钟内提供超声波。如果没有发生BBB开口，则可以增加峰值负压设置，但需要注意的是，较高的峰值负压会增加引起微出血的机会，我们在使用所述设置的0.65 MPa峰值负压设置下无法检测到微出血。根据抗体的抗原特异性，染色模式会有所不同。注射抗tau抗体时获得的染色模式如图 2所示。
该技术可以应用于一系列抗体，只要获得一致的BBB开口，就可以评估抗体与大脑中靶标的结合。扫描超声方法以可重复的方式在整个小鼠大脑中实现BBB的打开。
这种技术的局限性是，在小鼠活着时，不会观察到BBB开口的发生和范围。通过将钆造影剂的MRI成像纳入手术中可以克服这一限制，但这显着增加了手术的时间和成本。
这里描述的是单次超声处理和单次施用抗体方案，可用于确定可以达到多少抗体摄取增加，以及它们在交付后在大脑中的位置。该协议也可用于纵向研究，以评估抗体递送的治疗效果。在治疗研究中，该方案可以重复一周或更长时间的治疗间隔，以评估递送到大脑的抗体的治疗潜力。超声递送的抗体的治疗潜力可以在神经退行性疾病的转基因小鼠模型中评估。治疗效果的读数可能包括行为测试，以及病理蛋白水平的组织学和生物化学，例如tau，淀粉样蛋白β或突触核蛋白。
总之，我们已经概述了一种打开小鼠血脑屏障以提供荧光标记抗体的方法。评估神经退行性疾病治疗方法的研究人员将对这种方法感兴趣。

治疗性超声是一种新兴技术，旨在以非侵入性方式治疗脑部疾病，部分原因是通过促进治疗剂进入大脑1，2，3。由于只有一小部分针对脑部疾病的治疗性抗体被大脑吸收并保留在大脑中4，因此治疗性超声提供了增加其摄取和靶向参与的可能性5，6。
在我们的实验室中，我们正在开发神经退行性疾病的治疗方法，其中各种形式的抗体使用微气泡通过血脑屏障（BBB）递送。为了实现这一点，超声波通过颅骨施加到大脑的多个位置，使用我们称之为扫描超声波（SUS）7的扫描模式。超声能量、静脉注射微气泡和脑脉管系统之间的机械相互作用在给定的超声处理体积中瞬时分离BBB的紧密连接，允许抗体和包括治疗剂在内的其他货物有效地穿过这一屏障7，8，9.此外，超声已被证明可以促进抗体从间质性大脑摄取到脑细胞中，例如神经元，其中抗体分布在整个细胞体中，甚至进入神经炎过程5，10。
阿尔茨海默病的特征是淀粉样蛋白β和tau病理学11，并且可以使用许多动物模型来剖析致病机制并验证治疗策略。SUS方法，通过这种方法，超声波以顺序模式应用于整个大脑，当在几个治疗过程中重复时，可以减少淀粉样蛋白β沉积淀粉样蛋白前体蛋白（APP）突变小鼠大脑中的淀粉样蛋白斑块病理学，并激活占据淀粉样蛋白的小胶质细胞，从而改善认知功能7。用超声和微气泡打开BBB也会降低pR5，K3和rTg4510 tau转基因小鼠的tau病理学5，12，13。重要的是，虽然小胶质细胞去除细胞外蛋白沉积物，但SUS诱导的神经间病变的潜在清除机制之一是神经元自噬的激活12。
在这里，我们概述了一个实验过程，通过该过程制备荧光标记的抗体，然后与内部基于脂质的微泡混合，然后向麻醉小鼠进行逆眶注射。眶后注射是尾静脉注射的替代方案，我们发现尾静脉注射同样有效且易于重复执行。紧随其后的是将SUS应用于大脑。为了确定治疗性抗体摄取，处死小鼠，然后量化大脑中增加的抗体浓度。作为脑稳态变化的代表，小胶质细胞吞噬活性通过组织学和体积3D重建确定。
产生的数据表明，抗体的超声递送是治疗神经退行性疾病的潜在有吸引力的方法。该协议可以类似地应用于其他候选药物，以及模型货物，例如定义大小的荧光标记葡聚糖14。

<table><tbody><tr><td>1,2-distearoyl-sn-glycero-3-phosphocholine</td><td>Avanti</td><td>850365C</td><td></td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000]</td><td>Avanti</td><td>880128C</td><td></td></tr><tr><td>AlexaFluor 647 antibody labeling kit</td><td>Thermo Fisher</td><td>A20186</td><td></td></tr><tr><td>CD68 antibody</td><td>AbD Serotec</td><td>MCA1957GA</td><td>Use 1:1000 dilution</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>372978</td><td></td></tr><tr><td>Coulter Counter (Multisizer 4e)</td><td></td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Goat anti-rabbit IgG, Alexa Fluor 488</td><td>Thermo FIsher</td><td>A-11008</td><td>Use 1:500 dilution</td></tr><tr><td>Goat anti-rabbit IgG, Alexa Fluor 488</td><td>Thermo Fisher</td><td>A-11077</td><td>Use 1:500 dilution</td></tr><tr><td>head holder (model SG-4N, Narishige Japan)</td><td></td><td></td><td></td></tr><tr><td>Iba1 antibody</td><td>Wako</td><td>019-19741</td><td>Use 1:1000 dilution</td></tr><tr><td>Image analysis software</td><td>Beckman Coulter</td><td>#8547008</td><td></td></tr><tr><td>Isoflow flow solution</td><td>Beckman Coulter</td><td>B43905</td><td></td></tr><tr><td>Near infrared imaging system Odyssey Fc</td><td>Licor</td><td>2800-03</td><td></td></tr><tr><td>Octafluoropropane</td><td>Arcadophta</td><td>0229NC</td><td></td></tr><tr><td>Propylene Glycol</td><td>Sigma-Aldrich</td><td>P4347</td><td></td></tr><tr><td>TIPS (Therapy Imaging Probe System)</td><td>Philips Research</td><td>TIPS_007</td><td></td></tr><tr><td></td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

delivery of antibodies into the brain using focused scanning ultrasound

只有一小部分针对脑部疾病的治疗性抗体被大脑吸收。聚焦超声提供了通过血脑屏障（BBB）的短暂打开来增加抗体摄取和参与的可能性。在我们的实验室中，我们正在开发神经退行性疾病的治疗方法，其中使用微气泡将各种形式的抗体输送到BBB中，同时通过针对多个点的颅骨进行聚焦超声应用，我们称之为扫描超声（SUS）。微气泡和超声对血管的机械效应通过瞬时分离紧密连接来增加BBB的细胞旁转运，并增强囊泡介导的转运，使抗体和治疗剂能够有效地交叉。此外，超声还有助于将抗体从间质大脑摄取到脑细胞中，例如抗体分布在整个细胞体中的神经元，甚至进入神经炎过程。在我们的研究中，制备荧光标记的抗体，与内部制备的基于脂质的微气泡混合，并在SUS应用于大脑之前立即注射到小鼠中。然后量化大脑中增加的抗体浓度。为了解释正常脑稳态的改变，小胶质细胞吞噬作用可用作细胞标志物。产生的数据表明，抗体的超声递送是治疗神经退行性疾病的一种有吸引力的方法。

使用该协议，荧光标记的抗体被传递到大脑并且可以被检测到，以及小胶质细胞的激活。可以得出的结论是，使用聚焦超声和微气泡显着增强了抗体的大脑摄取，并且在扫描模式下使用时可以将抗体传递到小鼠的整个大脑或半球。 图1 显示了用于打开BBB的TIPS超声应用设备（标记的不同组件）。 图2 显示了库尔特计数器测量的尺寸和浓度的代表性结果，当微气泡正确产生时，应获得这些结果。为了容易地可视化递送，用远红色荧光染料标记抗体。使用红外扫描仪或使用荧光显微镜对大脑切片，可以在整个大脑或切片中轻松可视化大脑的抗体摄取。大脑切片在微观水平上显示荧光标记抗体的位置。扫描超声递送到荧光标记的抗tau抗体RN2N的海马体的代表性结果如图 3所示。为了观察SUS和抗体递送导致正常脑稳态的任何改变，一个读数是与吞噬作用相关的小胶质溶酶体含量。 图4 显示了使用Iba1和小胶质细胞溶酶体特异性标志物CD68对小胶质细胞的代表性染色，以确定小胶质细胞在抗体递送后是否变得更具有吞噬作用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61372/61372fig01.jpg"> 图 1:用于提供带有标记关键组件的超声的聚焦超声系统。 （A） 用作 8 mm 垫片的自制凝胶支架。（B）通过传感器查看目标如何与视觉焦点对齐（C）。 <a href="https://www.jove.com/files/ftp_upload/61372/61372fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61372/61372fig02.jpg"> 图2:内部制备的微气泡的质量控制测量。 （A）库尔特计数器设备，用于获取汇总统计量（B）和微气泡的大小分布（C）和体积分布（D）。 <a href="https://www.jove.com/files/ftp_upload/61372/61372fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61372/61372fig03.jpg"> 图3:使用SUS将荧光标记的抗体递送到大脑中。 （A）一种荧光标记的抗体片段，该抗体片段特异性于tau同种型自行递送，SUS本身以及联合治疗显示，当与SUS结合使用时，使用大脑一个半球的近红外荧光成像，超声半球对抗体的摄取增加。应用了查找表（LUT），以较暖的颜色显示任意单位的荧光强度更高。（B）荧光的定量在不减去仅SUS对照水平的情况下进行背景荧光。显示平均±扫描电镜。（C）海马神经元对荧光标记抗体的摄取增加，显示在大脑切片的低倍率和高倍率图像中。在联合治疗中，抗体分布到细胞体甚至树突中，如海马神经元所示。蓝色=DAPI，洋红色=抗体片段。比例尺:50 μm。 <a href="https://www.jove.com/files/ftp_upload/61372/61372fig03large.jpg" target="_blank">请单击此处查看此图的放大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61372/61372fig04.jpg"> 图4:小胶质细胞的代表性免疫荧光标记，显示小胶质细胞的预期形态，并使用图像分析软件以3D形式呈现。 小胶质细胞形态以绿色观察，CD68的水平和分布以红色观察。比例尺 10 μm. 绿色=Iba1， 红色=CD68. <a href="https://www.jove.com/files/ftp_upload/61372/61372fig04large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound at JoVE.com

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

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。

使用聚焦扫描超声将抗体输送到大脑中

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

delivery-antibodies-into-brain-using-focused-scanning

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.1K 次观看.  The University of Queensland. 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。

视频: Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

科研

医学

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

实时活体内多光子显微镜可可视化聚焦超声和微气泡处理，以提高血脑屏障通透性

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

生物工程

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 ...

神经科学

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Enhancing Drug Delivery to the Brain: MRI-Guided Ultrasound Techniques in Rats

Monitoring Blood-Brain Barrier Opening in Rats with a Preclinical Focused Ultrasound System

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic macromolecular agents to the brain. The integration of MR-guided low-intensity pulsed focused ultrasound (FUS) with microbubble pre-injection is a promising technique for non-invasive and non-toxic BBB modulation. MRI can offer superior soft-tissue contrast and various quantitative assessments, such as vascular permeability, perfusion, and the spatial-temporal distribution of MRI contrast agents. Notably, contrast-enhanced MRI techniques with gadolinium-based MR contrast agents have been shown to be the gold standard for detecting BBB openings. This study outlines a comprehensive methodology involving MRI protocols and animal procedures for monitoring BBB opening in a rat model. The rat model provides the added benefit of jugular vein catheter utilization, which facilitates rapid medication administration. A stereotactic-guided preclinical FUS transducer facilitates the refinement and streamlining of animal procedures and MRI protocols. The resulting methods are characterized by reproducibility and simplicity, eliminating the need for specialized surgical expertise. This research endeavors to contribute to the optimization of preclinical procedures with rat models and encourage further investigation into the modulation of the BBB to enhance therapeutic interventions in neurological disorders.

This study describes a combined magnetic resonance imaging (MRI) and low-intensity pulsed focused ultrasound (FUS) protocol, utilizing living rats with jugular vein catheterization to monitor blood-brain barrier (BBB) opening.

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic ...

Convection-Enhanced Delivery of Antibodies into a Mouse Brain

Source: Beffinger, M., et al. <a href="https://app.jove.com/v/59675">Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery.</a> J. Vis. Exp. (2019).
This video demonstrates a convection-enhanced delivery (CED) technique for antibody delivery into the mouse brain. The skull of an anesthetized mouse is exposed, and a burr hole is created at the target area for antibody delivery. A step catheter containing antibodies is inserted at the target site, and via an infusion pump, a pressure gradient is created to deliver antibodies at a controlled rate.

对流增强抗体向小鼠大脑的递送

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

An Ultrasound-Assisted Delivery of Antibodies in a Mouse Model

Source: Leinenga, G. et al., <a href="https://app.jove.com/v/61372">Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates a procedure of ultrasound-assisted antibody delivery in a mouse model. Retro-orbitally injected microbubbles oscillate under ultrasound, creating a transient disruption in the blood-brain barrier, permitting labeled antibodies to enter the brain.

小鼠模型中的超声辅助抗体递送

Focus Ultrasound Based Microbubble Mediated Blood-Brain Barrier Opening: A Technique to Create Localized Transient Openings in Blood-Brain Barrier of Mouse by Sonoporation

Source: Haumann, R. et al. <a href="https://www.jove.com/video/61269" target="_blank">A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents.</a> J. Vis. Exp. (2020)
This video describes a method of temporarily disrupting the blood-brain barrier (BBB) by creating transient openings using microbubble-mediated focused ultrasound (FUS). These transient local BBB openings can eventually be used to deliver larger-sized drug molecules directly into the brain parenchyma.

基于聚焦超声的微泡介导的血脑屏障开口:一种通过超声在小鼠血脑屏障中创建局部瞬时开口的技术

Source: Haumann, R. et al. A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in ...