这项研究部分得到了NSF EPSCoR研究基础设施基金（1632881）的支持。此外，这项研究部分得到了西维坦国际研究中心的支持，伯明翰，AL。作者感激地感谢阿拉巴马大学伯明翰小型动物成像共享设施赠款 [NIH P30 CA013148] 的服务和设施的使用。作者感谢拉吉夫·乔普拉的支持和指导。

所有实验程序均按照 UAB 机构动物护理和使用委员会 （IACUC） 准则进行。
1. 聚焦超声波驾驶设备设置
<ol><li>使用 50 Ohm 同轴 BNC 电缆将超声波传感器的输入连接到 RF 放大器的输出中，（2） RF 放大器对功能发生器输出的输入。</li>
	<li>将函数生成器模式设置为每秒一次的鼻窦爆裂，其工作周期为 1%。<ol>
<li>对于带 50 dB RF 放大器使用的 0.8 英寸焦距的阻尼 1 MHz 低功耗浸入式传感器，将启动设置设置为：1 MHz sine 波、1 V 峰值到峰值、10k 周期、1 s 间隔（或句点）。</li>
		<li>对于空气支持，1.1 MHz 大功率传感器，将初始设置设置为：1.1 MHz sine 波、50 mV 峰值到峰值、11k 周期、1s 间隔。 注意：除非传感器被淹没，否则不要操作传感器。操作时，不要将手或其他身体部位放在超声波聚焦处或触摸传感器面部。</li>
	</ol>
</li>
</ol>2. 聚焦超声台式设置
<ol><li>3D 打印立体声轴图框架和立体定向框架支架。</li>
	<li>使用握有PVC管道的夹子将传感器连接到XYZ定位器（图1a）。将夹子固定在 X 轴滑梯上，然后用翼螺母锁定。<ol>
<li>如果使用大功率超声波传感器，则使用匹配的磁铁将其连接到 PVC 管道上。确保一个磁铁有一个孔，另一个有一个匹配的突起。盖上PVC管的底部，用环氧树脂将其中一块磁铁附着在管道上（见图1b）。</li>
		<li>使用环氧树脂将第二块磁铁连接到大功率超声波传感器的顶部中心。确保它是在传感器的中心（图1c）。</li>
	</ol>
</li>
	<li>如果使用大功率超声波传感器，则制作一个用于取消 XYZ 定位器的指点。当超声波传感器连接到 XYZ 定位器时，此指尖表示传感器对焦中心的空间位置。用 18 G 针切出一个指针，作为传感器焦距的长度加上传感器的厚度（图 1d）。<ol>
<li>将环氧树脂涂抹到第三块磁铁上（这种磁铁也与PVC帽磁铁配对），并将其附着在指尖顶部。然后，指点将能够连接到PVC管上的磁铁上，用于XYZ空（图1d）。</li>
	</ol>
</li>
	<li>如果使用低功耗浸入式传感器，则 3D 打印指针和安装夹的文件。<ol>
<li>将低功耗浸入式传感器连接到PVC管道，将安装夹剪切到PVC管道上，并将传感器插入环（图1f）。</li>
	</ol>
</li>
	<li>将丙烯酸纸上切开的碎片粘在一起，使水浴成为水浴，这些碎块可以放在动物头顶上，高于立体定向框架（图1e）。<ol>
<li>在浴缸底部切开一个与动物头部大小差不多的开口。用聚酰胺胶带盖住水浴中的孔。 注意：必须小心，确保水不会泄漏周围的多酰胺胶带，因为它可以湿老鼠的毛皮，并导致体温过低。</li>
	</ol>
</li>
	<li>通过用 MRI 可见液体（如维生素 E 油）填充直径为 4 mm 的薄壳塑料或玻璃球，制成 MRI 纤维，并将其密封。将其放置在 3D 打印立体轴框架右侧的 MRI 支架中（图 2a）。</li>
	<li>牢固地将 3D 打印的框架支架固定在 XYZ 定位器上，以放置动物。将框架支架的旋端选项卡滑入 Y 轴轨道上的匹配通道，并使用固定螺钉（图 1h，红色箭头）进行固定。</li>
	<li>要驾驶 XYZ 定位器，请按照制造商的指示将 USB 安装到计算机上的串行转换器，并插入转换器。在 PC 上安装运行时间环境和电机控制器软件。<ol>
<li>通过在控制器软件前面板上的端口选择下拉控制中选择 USB 到串行转换器，确保在软件中选择正确的串行端口。使用 9 针串行交叉电缆（例如 RS232 空调制解调器电缆）将 USB 连接到串行转换器到继子电机控制器盒。</li>
		<li>运行控制器软件，以测试步进电机可以在软件控制下驱动。此步骤可能需要本地 IT 支持的帮助。</li>
	</ol>
</li>
</ol>3. 颅内定位程序
注：雄性斯普拉格道利大鼠体重250-350克用于这些实验。动物可以自由获得水和老鼠圈，并保持在12：12小时的光：黑暗循环。
<ol><li>将动物置于麻醉下（3%含氧异氟），并检查脚趾捏缺乏反应。然后插入如下所述的导管。<ol>
<li>用灯加热尾巴，使触脉更容易。小心不要使动物过热或烫伤尾巴。</li>
		<li>一旦动物睡着了（不回应脚趾捏），插入24G尾静脉导管，将用于提供微泡泡，埃文斯蓝色染料（EBD），加多布特罗MRI对比，如果使用MRI，和实验剂的兴趣。一旦静脉被击中，血液将充满护套，慢慢取出内针，同时将护套进一步推入静脉。 注意：如果第一次注射大鼠尾静脉，请参阅像斯图亚特和施罗德33 这样的指南。</li>
		<li>如果没有血流，慢慢将导管护套移出静脉，以测试针头可能戳穿了静脉。如果血液流动时，导管被稍微拉回，然后首先戳通过静脉和导管放置将需要重新启动在尾部的另一个位置，是旋转到以前的位置。</li>
	</ol></li>
	<li>用盐水填充导管插头，并在端口充满血液后立即将导管插头拧入导管端口的末端。小心地将实验室胶带缠绕在导管和尾部，使其保持原位。从顶部的一小块开始，朝方向工作，使导管插头的末端暴露在外。</li>
	<li>将麻醉线插入立体轴（图2a）上的麻醉连接器上，将嘴放在咬杆上，引导耳栏进入两个耳道，然后拧紧固定螺丝，将动物头部固定在镜框内。确保动物头部的安全和水平。</li>
	<li>将动物移入核磁共振成像（MRI）床，并将麻醉线连接到鼻锥。在此协议中使用了 9.4 T 小孔动物 MRI。</li>
	<li>收集冠状和轴向T2加权图像，捕捉整个大脑以及MRI信托（图2b）进行坐标测量。提供当地的核磁共振成像物理学家或技术人员以下信息，以便他们能够构建 MRI 协议。<ol>
<li>对于日冕图像（图2b顶部），使用以下参数：图像数量：27，宽度：62.2毫米，高度：62.2毫米，深度：37.97毫米，Voxel大小：0.24 x 0.24 x 1.41毫米3。</li>
		<li>对于轴向图像（图2b底部），使用以下参数：图像数量：13，宽度：61.47毫米，高度：53.81毫米，深度：16.7毫米，Voxel大小：0.41 x 0.21 x 1.29毫米3。 注：只要平面分辨率的日冕接近0.25毫米，图像覆盖整个大脑和受体，就没有必要有这些确切的参数。</li>
	</ol></li>
	<li>通过记录从 MRI 受托到以 FUS 为目标的大脑区域的距离，从上述图像中收集坐标测量结果。<ol>
<li>在扫描仪上，在步骤 3.5 中收集的日冕图像上，找到受体最大的图像，指示受体的中心。在介质/横向方向和右心室方向（图2b，顶部）记录从受体顶部到对毫米感兴趣的大脑区域的距离（MRI软件将有一个刻度或点测量工具，咨询当地的MRI技术或物理学家如何做到这一点）。</li>
		<li>在扫描仪上，在步骤 3.5 中收集的轴向图像上，找到显示受体顶部的图像，并测量从受体中心到目标大脑区域在指端/腹向和中间/横向方向的距离（图 2b，底部）。</li>
		<li>比较两个介质/横向测量，如果它们是不同的使用平均值。这些坐标测量将在第 4.3 步晚些时候使用，用于用 XYZ 定位器引导 FUS 焦点到目标大脑区域。</li>
	</ol></li>
	<li>收集 MRI 预扫描图像。将这些图像与 FUS BBB 打开后收集的图像进行比较（图 4）。T1 加权图像稍后将用于可视化 BBB 开口，T2 加权图像稍后将用于确保 FUS 治疗34后不会发生组织损伤。<ol>
<li>对于 T1 加权轴向图像，请使用以下参数：宽度：30 毫米，高度：51.2 毫米，深度：3.0 毫米，voxel 大小：0.23 x 0.2 x 0.23 mm3，图像数量：13。</li>
		<li>对于 T2 加权轴向图像，请使用以下参数：宽度：30 毫米，高度：51.2 毫米，深度：2.6 毫米，voxel 大小：0.2 x 0.2 x 0.2 mm3，图像数量：13。</li>
		<li>对于T1加权日冕图像，请使用以下参数：宽度：30毫米，高度：30毫米，深度：27毫米，体型：0.16 x 0.16 x1毫米3，图像数量：27。</li>
		<li>对于 T2 加权日冕图像，请使用以下参数：宽度：30 毫米，高度：30 毫米，深度：27 毫米，体型：0.12 x 0.12 x 1 mm3，图像数量：27。 注：与第 3.5 步一样，这些成像参数不必与列出的参数完全相同。这些图像的 FOV 更小，分辨率也高于第 3.5 步收集的图像。</li>
	</ol></li>
	<li>将动物保持在立体轴框架中，快速将动物从 MRI 床运送到台面 FUS 设置。确保动物在麻醉作用下保持睡眠以进行转移。</li>
	<li>对于更长的传输时间，使用足够大的盒子进行传输，以适应动物和框架。麻醉塞仍然连接，将动物和框架放在盒子内，让多余的异氟化物填充盒子几分钟。拔下麻醉线，快速转移。</li>
</ol>4. 聚焦超声波手术
<ol><li>到达 FUS 台面设置后，立即将麻醉线插入鼻锥，并继续用氧气运行 1.5-3% 异氟化物。尽快这样做，以避免动物醒来。</li>
	<li>将框架滑入框架支架，并牢牢地将其捕捉到位。用剪子剃动物的头。刷掉多余的头发，在头皮上涂抹脱发霜。让我们坐3分钟，用水和纱布擦去。</li>
	<li>如果 MRI 不可定位，请使用标准（非 3D 打印）立体定向框架，通过触摸指尖到 bregma（为此需要头皮切口）并取消软件或记录坐标，将指点位置无效。通过单击软件中的 向上 50 个按钮并将指头换成传感器，将 XYZ 车厢向上移动 50 mm。根据之前描述的35个鼠脑地图集，使用软件中的步进按钮移动到所需的大脑坐标。如果使用此方法而不是 MRI，请跳到第 4.6 节。</li>
	<li>如果使用 MRI 指导，则将指点连接起来，并将指头移动到 MRI 受体的位置（图 1d，g）。将指头放置在 MRI 支架的顶部和中心（为指点提供了支架顶部的一个小孔）。单击空位按钮，该按钮是计算 MRI 图像中所有距离的点。</li>
	<li>取下指头，将定位器移动到中/横向坐标和旋翼/腹地坐标。通过按 下向上 50 按钮将定位器抬起来，以便放置水浴和超声波凝胶。如果到达 Z 轴旅行的顶部，则无效位置将失效。在添加传感器后，将设置正向/腹腔坐标。</li>
	<li>将超声波凝胶涂在动物的头皮上，将水浴放在动物身上，将聚酰胺胶带窗口压在凝胶上。确保超声波凝胶中没有气泡。</li>
	<li>在水浴中加满脱气水。</li>
	<li>如果使用大功率传感器，降低定位器，使磁铁就在水面上。将传感器连接到定位器上，小心地将传感器以角度放入水中，以防止气泡困在面部下方并连接磁铁。</li>
	<li>如果使用低功耗浸入式传感器，则将定位器降低到传感器剪辑正上方的水中。然后通过以一个角度缓慢地将其降低到水中，将传感器夹在原位，以防止气泡被困在脸部下面。 注意：透明浴池在传感器表面下寻找气泡时很有帮助。</li>
	<li>将定位器降低到右/室坐标。</li>
	<li>打开RF电源放大。</li>
	<li>通过将针尖插入导管插头并注射，将 1 mL/kg 的 3% 埃文斯蓝色染料 （EBD） 注入。允许它循环5分钟。</li>
	<li>用气泡摇床剧烈摇动微泡，激活微泡。<ol>
<li>在 0.2 mL 盐水中准备 30 μL/kg 微泡（气泡凝结 1.2 x10 10/mL）的 5 倍剂量，以考虑 2 FUS 处理和 18 G 有翼输液集中的管子。例如，如果大鼠体重为 200 克，则用 1 mL 盐水中的 30 μL 微泡填充含有 18 G 针尖的注射器。 注意：请务必使用 18 G 针尖，同时使用有翼输液装置进行取药和注射。</li>
		<li>多次倒置注射器以获得微泡的均匀分布。然后连接并填充有翼输液集。将注射器放置在输液泵上，并设置输液泵以 6 mL/h 的速度提供 0.2 mL。这将在 2 分钟的 FUS 暴露下提供微泡的缓慢输注。</li>
		<li>将有翼针插入导管插头。</li>
	</ol></li>
	<li>首先，运行输液泵，等待3s，然后通过按下功能发生器上的输出启用按钮（标记为"在材料表中的功能发生器上"）启动FUS处理。每个区域重复两次，中间5分钟，让微泡清除。<ol>
<li>当输液泵在 2 分钟内停止时，再次按下功能生成器 上的打开按钮 以停止 FUS 处理。</li>
		<li>等待 5 分钟，微泡清除。然后开始输液和第二次FUS治疗。</li>
		<li>在第二次 FUS 治疗后，立即注射加多布特罗对比度（如果使用 MRI）和感兴趣的代理，例如病毒颗粒。所有代理的总交付量不应超过 5 mL/kg。 注：利息代理的交货时间（例如，FUS BBB 开张前后）可能因所使用的代理而异。</li>
	</ol></li>
	<li>关闭 RF 电源放大，并立即将动物运回 MRI。</li>
</ol>5. BBB 开口的 MRI 确认
<ol><li>如果MRI不可用，请跳到第6节，使用EBD表达式确认BBB开口。</li>
	<li>将动物放回 MRI 床上，位置与步骤 3.7 完全相同，并插入麻醉线。</li>
	<li>收集MRI后扫描与第3.7步中使用的相同成像参数，以可视化BBB开口区域的加多布特罗MRI增强（图3b，e）。</li>
</ol>6. 注入和组织收集
<ol><li>用冷4%的正式素灌输动物，直到血液完全清除。</li>
	<li>去除大脑，并在4°C的4%正式或PFA过夜。接下来，将大脑置于30%的蔗糖溶液中，直到大脑下沉（约2-3天）。最后，在液氮或干冰上闪光冻结，并储存在-80°C，直到低温。</li>
	<li>冻结在OCT的大脑，并采取低温。</li>
	<li>用于荧光显微镜的修复和盖片部分。EBD 激发峰值为 470 和 540 nm，排放峰值为 680 nm。带 DAPI 安装介质的封面滑动，以可视化整体细胞形态。</li>
</ol>

作者没有什么可透露的。

在这里，我们描述了一个台面方法微泡协助FUS BBB打开与替代方法，包括，两个不同的传感器和方法的颅内瞄准有和没有MRI指导。目前，为了在实验室中建立 MRI 引导的 FUS BBB 开口，可以选择购买具有高度标准化和可重复效果的设备，并配备用户友好的界面。然而，许多实验室没有为此类仪器的成本做好准备。因此，此协议的主要目标是提供任何实验室都可以建立的起点，以建立他们在该技术方面?...

尽管在基础神经科学方面有许多发现，但神经发育和神经退行性疾病的新疗法数量仍然相对有限。对神经紊乱中涉及的基因、分子和细胞回路的更深入的了解表明，用目前的技术3，人类无法实现有希望的治疗方法。有效的治疗往往受到大脑渗透和特定地点4，5，6，7，8的需要的限制。然而，现有的局部药物输送到特定大脑区域的方法（例如，通过注射或针管分娩）是侵入性的，需要在头骨9中打开。这种手术的侵入性防止了将局部分娩的常规使用到人脑中。此外，组织损伤和由此产生的炎症反应是无处不在的混淆的基本和前科研究，依靠颅内注射10。非侵入性地通过血脑屏障（BBB）输送制剂并将其靶向特定大脑区域的能力可能会对神经系统疾病的治疗产生巨大影响，同时为术前研究提供强大的研究工具。
在BBB上以最小的组织损伤进行定向传输的方法之一是颅内聚焦超声波（FUS）和微泡，以聚焦和瞬时打开BBB 11、12、13、14、15、16。FUS BBB 的开放最近因神经退行性疾病的治疗而备受关注， 中风和胶质瘤通过本地化治疗目标大脑区域，如神经营养因子17，18，19，基因疗法20，21，22，抗体23，神经递质24，和纳米粒子25，26，27，28，29。FUS BBB 具有广泛的应用范围和非侵入性30、31，是常规立体轴心颅内注射的理想替代品。此外，由于它目前在人类中使用30，32，使用这种技术的术前研究可以被认为是高度转化的。然而，由于缺乏可访问性，FUS BBB的开放尚未成为基础科学和学前研究中广泛确立的技术。因此，我们为 FUS BBB 开启的台面方法提供了详细的协议，作为有兴趣建立此技术的实验室的起点。
这些研究要么使用大功率空气支持的FUS特定超声波传感器，要么使用低功率阻尼聚焦超声波浸入式传感器进行。传感器由专为活性负载设计的 RF 功率放大器和标准台面功能发生器驱动。这些物品的详情可在 材料表中找到。

<table border="0" cellpadding="0" cellspacing="0" style="width:1437px;" width="1437">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Bubble shaker</td>
			<td style="width:240px;">Lantheus Medical Imaging</td>
			<td style="width:197px;">VMIX</td>
			<td style="width:729px;">VIALMIX, actiation device used to activate Definity microbubbles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Catheter plug/ Injection cap</td>
			<td>SAI infusion technologies</td>
			<td>Part Number: IC</td>
			<td style="width:729px;">Catheter plug/ Injection cap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Evans blue dye</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:197px;">E2129-10G</td>
			<td style="width:729px;">Evans blue dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Function generator</td>
			<td style="width:240px;">Tektronix</td>
			<td style="width:197px;">AFG3022B</td>
			<td>Dual channel, 250MS/s, 25MHz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FUS transducer, 1.1MHz</td>
			<td style="width:240px;">FUS Instruments</td>
			<td>TX-110</td>
			<td>1 MHz MRI-compatible spherically focused ultrasound transducer with a hydrophone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Heating pad for Mice and Rats</td>
			<td style="width:240px;">Kent Scientific</td>
			<td style="width:197px;">PS-03</td>
			<td>Heating pad- PhysioSuite for Mice and Rats</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Infusion pump</td>
			<td style="width:240px;">KD Scientific</td>
			<td style="width:197px;">780100</td>
			<td>KDS 100 Legacy Single Syringe Infusion Pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Kapton tape</td>
			<td style="width:240px;">Gizmo Dorks</td>
			<td>https://www.amazon.com/dp/B01N1GGKRC/ 
			ref=cm_sw_em_r_mt_dp_U_GbR7Db56HKD91</td>
			<td>Gizmo Dorks Kapton Tape (Polyimide) for 3D Printers and Printing, 8 x 8 inches, 10 Sheets per Pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low power immersion transducer, 1MHz</td>
			<td style="width:240px;">Olympus</td>
			<td>V303-SU</td>
			<td>Immersion Transducer, 1 MHz, 0.50 in. Element Diameter, Standard Case Style, Straight UHF Connector, F=0.80IN PTF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Magnet sets</td>
			<td style="width:240px;">WINOMO</td>
			<td>https://www.amazon.com/dp/B01DJZQJBG/ 
			ref=cm_sw_em_r_mt_dp_U_JYQ7DbM32E5QC</td>
			<td>WINOMO 15mm Sew In Magnetic Bag Clasps for Sewing Scrapbooking - 10 Sets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RF amplifier</td>
			<td style="width:240px;">E&#38;I</td>
			<td style="width:197px;">A075</td>
			<td>75W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tail vein catheter</td>
			<td style="width:240px;">BD</td>
			<td>382512/ Fisher Item: NC1228513</td>
			<td>24g BD Insyte Autoguard shielded IV catheters (non-winged)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ultrasound contrast microbubbles</td>
			<td>Lantheus Medical Imaging</td>
			<td style="width:197px;">DE4, DE16</td>
			<td>DEFINITY (Perflutren Lipid Microsphere)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ultrasound gel</td>
			<td style="width:240px;">Aquasonic</td>
			<td>https://www.amazon.com/dp/B07FPQDM4F/ 
			ref=cm_sw_em_r_mt_dp_U_D6Q7Db3J9QP7P</td>
			<td>Ultrasound Gel Aquasonic 100 Transmission 1 Liter Squeeze Bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Winged infusion sets, 22ga.</td>
			<td style="width:240px;">Fisher Healthcare</td>
			<td>22-258087</td>
			<td>Terumo Surflo Winged Infusion Sets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">motor controller software</td>
			<td style="width:240px;">N/A</td>
			<td>N/A</td>
			<td style="width:729px;">custom software written in LabView for controlling the Velmex motor controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">runtime environment for the motor controller software</td>
			<td style="width:240px;">National Instruments</td>
			<td>LabView runtime engine version 2017 or better</td>
			<td>https://www.ni.com/en-us/support/downloads/software-products/download.labview.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3 axis Linear stage actuator (XYZ positioner)</td>
			<td style="width:240px;">Velmex</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bolts</td>
			<td style="width:240px;">Velmex</td>
			<td>MB-1</td>
			<td>BiSlide Bolt 1/4-20x3/4&#34; Socket cap screw (10 pack), Qty:3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">motor controller</td>
			<td style="width:240px;">Velmex</td>
			<td>VXM-3</td>
			<td style="width:729px;">Control,3 axis programmable stepping motor control, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting cleats</td>
			<td style="width:240px;">Velmex</td>
			<td>MC-2</td>
			<td>Cleat, 2 hole BiSlide, Qty:6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting cleats</td>
			<td style="width:240px;">Velmex</td>
			<td>MC-2</td>
			<td style="width:729px;">Cleat, 2 hole BiSlide, Qty:2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">usb to serial converter</td>
			<td style="width:240px;">Velmex</td>
			<td>VXM-USB-RS232</td>
			<td style="width:729px;">USB to RS232 Serial Communication Cable 10ft, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">x-axis linear stage</td>
			<td style="width:240px;">Velmex</td>
			<td>MN10-0100-M02-21</td>
			<td style="width:729px;">BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">x-axis stepper motor</td>
			<td style="width:240px;">Velmex</td>
			<td>PK266-03A-P1</td>
			<td>Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">y-axis linear stage</td>
			<td style="width:240px;">Velmex</td>
			<td>MN10-0100-M02-21</td>
			<td>BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">y-axis stepper motor</td>
			<td style="width:240px;">Velmex</td>
			<td>PK266-03A-P1</td>
			<td style="width:729px;">Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">z-axis damper</td>
			<td style="width:240px;">Velmex</td>
			<td>D6CL-6.3F</td>
			<td style="width:729px;">D6CL Damper for Type 23 Double Shaft Stepper Motor, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">z-axis linear stage</td>
			<td style="width:240px;">Velmex</td>
			<td>MN10-0100-M02-21</td>
			<td style="width:729px;">BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">z-axis stepper motor</td>
			<td style="width:240px;">Velmex</td>
			<td>PK266-03B-P2</td>
			<td style="width:729px;">Vexta Type 23T2, Double Shaft Stepper Motor, Qty:1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3D printable files</td>
			<td style="width:240px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Immersion transducer mount and pointer</td>
			<td></td>
			<td style="width:197px;"></td>
			<td>https://www.tinkercad.com/things/cRgTthGXSRq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stereotaxic frame</td>
			<td></td>
			<td style="width:197px;"></td>
			<td>https://www.tinkercad.com/things/ilynoQcdqlH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stereotaxic frame holder</td>
			<td></td>
			<td style="width:197px;"></td>
			<td>https://www.tinkercad.com/things/aZNgqhBOHAX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">9.4T small bore animal MRI</td>
			<td style="width:240px;">Bruker</td>
			<td style="width:197px;">Bruker BioSpec 94/20</td>
			<td>ParaVision version 5.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">AAV9-hsyn-GFP</td>
			<td style="width:240px;">Addgene</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Cream hair remover</td>
			<td style="width:240px;">Church &#38; Dwight</td>
			<td style="width:197px;"></td>
			<td>Nair cream</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">gadobutrol MRI contrast agent</td>
			<td style="width:240px;">Bayer</td>
			<td style="width:197px;"></td>
			<td>Gadavist (Gadobutrol, 1mM/mL)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stereotactic frame</td>
			<td style="width:240px;">Stoelting</td>
			<td style="width:197px;">#51500</td>
			<td>not MRI compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">turnkey FUS delivery device</td>
			<td style="width:240px;">FUS Instruments</td>
			<td style="width:197px;">RK-300</td>
			<td style="width:729px;">ready to use MRI compatible FUS for rodents</td>
		</tr>
	</tbody>
</table>

a benchtop approach to the location specific blood brain barrier opening using focused ultrasound in a rat model

立体税手术是局部药物和基因输送到啮齿动物大脑的黄金标准。与系统传递相比，这项技术有许多优点，包括精确定位到目标大脑区域和减少目标外的副作用。然而，立体轴手术是高度侵入性的，它限制了其转化功效，需要很长的恢复时间，并在瞄准多个大脑区域时提供挑战。聚焦超声波 （FUS） 可与循环微泡结合使用，在毫米大小的区域瞬时打开血脑屏障 （BBB）。这允许系统交付的代理的颅内本地化，这些代理通常不能穿过 BBB。这项技术提供了立体轴手术的非侵入性替代方法。然而，由于获得设备和标准化方法的机会有限，该技术迄今尚未在神经科学实验室得到广泛采用。该协议的总体目标是为 FUS BBB 开启 （BBBO） 提供一种台面方法，这种方法经济实惠且可重复，因此很容易被任何实验室采用。

在这里，我们演示了用微泡聚焦超声波可以使用上述低功耗浸入传感器（图3）和FUS传感器（图4）指定的参数诱导本地化BBB开口。首先，在早期实验中，低功耗浸入式传感器被瞄准一个大脑半球（图3b）或中位（图3a）。动物在2小时后被牺牲与注入（图3a）或没有注入（?...

Watch this Scientific Journal Video about A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model at JoVE.com

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

用微泡剂聚焦超声波可以聚焦和瞬时打开血脑屏障。这项技术已被用于通过血脑屏障提供广泛的制剂。本文提供了一个详细的协议，本地化交付给啮齿动物的大脑有或没有MRI指导。

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-benchtop-approach-to-location-specific-blood-brain-barrier-opening

Research

JoVE Journal

Neuroscience

4.5K 次观看.  University of Alabama at Birmingham. 用微泡剂聚焦超声波可以聚焦和瞬时打开血脑屏障。这项技术已被用于通过血脑屏障提供广泛的制剂。本文提供了一个详细的协议，本地化交付给啮齿动物的大脑有或没有MRI指导。

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

科研

神经科学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

研究病原体或药物交叉障碍及其与大脑相互作用的人血脑界面模型

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

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 ...

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

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 ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

源自人类多能干细胞的 3D 血脑屏障模型

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

作者聚焦：使用体外血脑屏障模型研究阿尔茨海默病的个性化方法 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

体外血脑屏障重建，模拟和治疗神经系统疾病

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...