Name: high-resolution confocal imaging of the blood-brain barrier: imaging, 3d reconstruction, and quantification of transcytosis
Uploaded: 2017-11-16T14:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis at JoVE.com

车的工作得到了罗氏博士后奖学金 (2014-2017) 的支持。

这项研究的道德批准由瑞士联邦食品安全和兽医局提供。所有动物实验都是严格遵守《瑞士联邦法令》关于动物保护和福利的, 以及根据《实验室动物护理国际评估和认可协会 (AAALAC) 规则》进行的. 
 1. 大脑自由浮动部分的生成 <ol> <li> 以确保最佳的取样质量, 在灌注的当天为磷酸盐缓冲盐水 (PBS) 制备2% 甲醛 (粉煤灰) 的新溶液. 
	注意: 粉煤灰是适度毒性皮肤接触和可能致癌物。使用丁腈手套处理粉煤灰, 并准备在化学油烟罩的解决方案。
	<ol> <li> 每只动物准备60毫升的煤灰溶液. </li>
		<li> 通过增加 180-200 和 #181 的 pH 值, 将粉煤灰转化为溶液; 每100毫升 PBS 的 5 M KOH 溶液, 并将溶液加热至60和 #176; C. 
		注: 不应使用氢氧化钠, 因为它对组织保存后的固定有负面影响. </li>
		<li> 让溶液冷却到室温, 并使用 HCl 将 pH 值降低到 7.4. 使用滤纸筛选解决方案 (请参阅 材料表 ). </li>
	</ol> </li> <li> 通过 transcardial 灌注进行完整的鼠标固定, 如前所述 15 进行了一些修改。首先, 冲洗血液从血管使用20毫升 PBS 然后灌注与40毫升2% 粉煤灰. </li>
	<li> 从头骨中删除大脑, 如前所述 15 . </li>
	<li> 浸泡一个新鲜的2% 粉煤灰灌流和 #160; 20 毫升2% 粉煤灰 7 h 在4和 #176; C post-fixation. 
	注: 不建议增加粉煤灰中的孵育, 因为它能防止细胞内结构的检测. </li>
	<li> 用 ice-cold PBS 广泛冲洗大脑. </li>
	<li> 在琼脂糖中嵌入固定的大脑. 
	<ol> <li> 使用微波加热在 PBS 中制备3% 琼脂糖溶液。轻轻旋转的解决方案降温, 但避免凝固. </li>
		<li> 在塑料容器中浸泡到琼脂糖溶液中之前, 先将 PBS 周围的多余部分晾干。在琼脂糖内旋转大脑以消除气泡。让琼脂糖凝固, 冷却在冰上. </li>
		<li> 小心地从塑料容器中取出琼脂糖块, 用刀片将大脑周围的立方体切掉。使用氰胶水将大脑安装在 vibratome 标本架上 (请参见 材料表 ) (请参阅 材料表 )。在进行切片之前, 允许有足够的时间来固化胶水. </li>
	</ol> </li> <li> 将标本持有者的大脑转移到填充 PBS 的 vibratome 缓冲托盘中。使用 vibratome 100 和 #181; m 脑切片 (矢状或冠状)。收集先前充满 PBS 的6井盘中的脑部. </li>
	<li> 在完成脑切片后, 小心地移除 pbs, 用1:1 的 pbs/甘油溶液代替. </li>
	<li> 在-20 和 #176 中存储 PBS/甘油中的部分; C. </li>
</ol> 2。免疫荧光染色的细胞或器官的标记 <ol> <li> 将大脑部分小心地转移到以前充满500和 #181 的24孔板的井中; 每井的 PBS。该节将保持在同一井, 直到程序结束. </li>
	<li> 用500和 #181 冲洗该节两次; PBS 的 L 在温和的鼓动下5分钟. </li>
	<li> 移除 PBS 并执行同时阻断和性的脑切片. 
	<ol> <li> 在 PBS 中准备一个0.3% 卫一 x 和10% 驴血清的阻断和性溶液. 
		注: 驴血清可被山羊血清替代, 以匹配寄主物种的特定二次抗体使用. </li>
		<li> 在室温下以250和 #181 的方式孵育剖面; 在温和的搅动下, 性溶液为1小时. </li>
	</ol> </li> <li> 删除解决方案, 并在适当的稀释中添加含有5% 驴血清和主抗体的 PBS 解决方案 (如 、1:100 到1:1、000)。在4和 #176 夜间孵育; 在温和的鼓动下. 
	注意: 有关使用此协议成功标记 NVU 的不同单元格类型的抗体列表, 请参见 材料表 。主要抗体的最佳的稀释必须经验主义地被确定。对于不同抗原的同时标记, 在同一溶液中稀释所有主抗体。所有的主要抗体必须在不同的物种中提出。为了提高抗体的穿透能力, 可以在4和 #176 中孵育72小时的样品; C. </li>
	<li> 在室温下温和搅拌的情况下, 在 PBS 中移除抗体溶液和洗涤部分三次, 以10分钟。删除 pbs 并添加250和 #181; L pbs 溶液含有5% 驴血清和适当的 species-specific 荧光标记的二级抗体。在室温下孵育1小时在温和的鼓动下的部分. 
	<ol> <li> 有关与此协议一起使用的次要抗体列表, 请参见 材料表 。
	</ol> </li> <li> 在室温下温和搅拌下, 在 PBS 中删除抗体溶液并清洗三次, 10 分钟. </li>
	<li> 删除 PBS 并添加250和 #181; 4 和 #39; 6-diamidino-2-吲 (DAPI) 解决方案与 #160; 最后集中1和 #181; g/毫升。在室温下孵育10分钟, 在温和的鼓动下。删除 DAPI 解决方案和洗涤部分3次, 5 分钟与 PBS. </li>
	<li> 在接合显微镜幻灯片 ( 例如, 组织键玻璃幻灯片) 上安装大脑部分。小心地在幻灯片上删除多余的 PBS。添加一滴安装介质 (见材料表) 在大脑部分的顶部, 并小心地覆盖它与0.17 毫米 (No. 1.5) 硼硅酸盐玻璃片. 
	<ol> <li> 将示例存储在4和 #176; C 在执行图像获取之前保护不受光照. </li>
	</ol> </li> </ol> 3。血脑屏障的高分辨率共聚焦成像 <ol> <li> 使用合适的激光扫描共聚焦显微镜 (参见材料 表 ) 用激光线在405、488、561和 633 nm 进行图像采集, 以激发荧光在蓝色, 绿色, 橙色, 和红色区域的光频谱。对于图像采集, 使用一个数字孔径为1.4 的63X 油目标. </li>
	<li> 在采集菜单中控制显微镜的软件, 设置图像采集参数。在 #8216 的下拉菜单中; 图像大小和 #8217; 选择一个 1024年 x 1024 像素的值。通过调整 #8216 的值, 将像素大小修改为介于200和 300 nm 之间的值; 缩放和 #8217; 菜单. #8221; 注意: 对于细胞内结构的分析, 将像素大小减少到 75 nm。此图像大小设置极大地增加了捕获时间, 并可以减少到 512 x 512 像素, 通过成像较小的视场来减少捕获时间. </li>
	<li> 在 #39; 获取速度和 #39; 下拉菜单中, 选择一个值 400 Hz, 即 每秒400行。在和 #39; 系统设置和 #39;菜单中, 选择 #39;P ixel 深度和 #39; 下拉菜单, 并将该值更改为12位. </li>
	<li> 在显微镜软件中, 在 #39; 获取和 #39; 选项卡上, 切换用于顺序帧获取的选项。通过修改 #39;P inhole 和 #39; 菜单中的值, 将光学剖面厚度设置为每个通道的0.75 和 1 #181; m. </li>
	<li> 在面板中进行荧光激发, 激活所需的激光以最佳激发样品中的荧光, 例如 488 nm 的激光线为绿色发射的荧光. 
	注意: 使用荧光频谱查看器 (请参阅 材料表 ) 选择足够的激发激光器. </li>
	<li> 在用于荧光检测的面板中, 移动滑块以选择将在每个通道中测量的波长, 例如, 在510和 550 nm 之间进行绿色发射的荧光. 
	注意: 使用荧光频谱查看器 (请参阅 材料表 ) 选择适当的探测波长. </li>
	<li> 添加一滴浸入式油, 其折射率1.52 在片的顶部与大脑部分, 以匹配的折射率的玻璃片和目标。将样品放在显微镜下, 打开 epifluorescent 灯, 用显微镜望远镜对样品进行可视化. </li>
	<li> 使用显微镜支架上的按钮, 更改过滤轮以选择适合于可视化 DAPI 染色核的过滤器。使用粗糙的焦点, 使信号从 DAPI 染色的细胞核进入焦点. </li>
	<li> 使用显微镜支架上的按钮, 更改滤镜以可视化血管标记 (例如胶原或 CD31) 的信号, 并将视场集中在单个毛细管段上. </li>
	<li> 按下按钮以启动实时扫描模式。在扫描过程中, 调整每个通道的增益和激光强度, 以最大化图像的动态范围, 避免像素饱和. 
	注意: 使用查找表来标记饱和像素以直观地评估饱和。为了避免信号饱和, 请使用预期具有最高荧光信号的样品来调整设置. </li>
	<li> 将行的平均值设置为 2. 
	注意: 当使用较高的采集速度时, 增加此值以降低噪音. </li>
	<li> 建立开始和结束部分, 用于执行横跨毛细管的整个体积的光学 z 堆叠。使用0.45 和 #181 的步骤大小; m. 
	注意: 如果图像将用于后续量化, 请为完整的数据集保留相同的获取设置. </li>
	<li> 对于量化, 从至少三不同的小鼠中每节获得10到 20 z 堆栈进行统计比较. 
	注: 在同一时段内获取完整的数据集, 以减少样品漂白和激光强度波动引起的变化. </li>
</ol> 4。神经血管单元的图像处理与3D 重建 <ol> <li> (可选) 若要增加所获得的 z 堆栈的轴向分辨率, 请使用适当的软件执行图像数据集的反卷积 (请参见 材料表).
	<ol> <li> 在反卷积软件中, 将设置更改为执行和 #34; 盲 #34; 反卷积 ( 即 , 使用自适应点扩展函数). </li>
		<li> 在反卷积软件设置中, 切换用于背景删除的选项; 此选项将确定图像堆栈中最小的亮度值, 并将其从图像中所有像素的强度值中减去. </li>
		<li> 在反卷积软件设置中, 切换缩放强度的选项, 并将其大小调整为16位深度; 这些选项将保留图像的原始强度值和动态范围. </li>
		<li> 在 #39 的菜单中; 折射率和 #39;, 将值设置为1.52。在和 #39; 设置波长和 #39; 菜单, 选择每个图像通道的发射值。例如, 为绿色发光的荧光选择 520. </li>
	</ol> </li> <li> 使用适合于3维数据集的可视化和分析的软件打开图像数据集 (请参见 材料表 )。在图像分析软件中, 按下 #34; 超过和 #34; 按钮以呈现3D 体积的毛细管. </li>
	<li> 在图像分析软件中按下和 #34; 添加新的曲面和 #34; 按钮, 以分割毛细管表面。使用底部箭头在 "曲面创建向导" 中的步骤之间移动. 
	在和 #39 中的 <ol> <li>; 创建和 #39; 选项卡上, 将源通道设置为带有毛细管标记的通道, 例如胶原, 并将曲面区域详细信息设置为值0.5 和 #181; m. 
		注意: 后一种选项将高斯滤镜应用于图像, 并确定曲面的平滑度. </li>
		<li> 在 #39; 创建和 #39; 选项卡上, 切换为绝对强度阈值的选项. 
		注意: 此选项将根据图像中所选通道的绝对强度生成掩码来创建曲面. </li>
		<li> 在曲面创建向导的下一步中, 通过将滑动窗口移过强度直方图来调整该曲面的较低强度阈值, 直到呈现的掩码覆盖图像中的整个毛细管。根据经验为每个数据集确定此参数的值范围. </li>
		<li> 在曲面创建向导的最后一步, 单击下拉菜单下的 #34; 筛选类型和 #34; 选择选项和 #39; 体和 #39 的数量;。将最小体数设置为介于1.0e5 到2.0e5 之间的值. 
		注意: 此选项将排除具有少量体的曲面, 例如, 框架边缘的小段毛细血管. </li>
		<li> 完成曲面创建向导并通过单击和 #34 保存创建参数; 记住参数和 #34; 在 "曲面" 菜单的 "重建" 选项卡中. </li>
		<li> 通过加载用于第一个图像的参数集来重复整个成像数据集的过程。调整每个图像的强度、阈值和最小体数的参数, 分别解释背景强度和毛细管形态的差异. </li>
	</ol> </li> <li> 要分割胞内水疱结构 (如 , 免疫球蛋白填充的体), 首先创建一个新的通道, 它只包括毛细管内部的荧光信号. 
	注意: 此过程将通过在步骤 4.3 <ol> <li> 中创建的毛细管表面创建掩码来定义感兴趣的区域, 方法是选择 #39; 编辑和 #39; #39 中的菜单; 毛细管表面和 #39; 面板。下/#39; 掩码属性和 #39;, 单击并 #34; 掩码 #34;。选择包含胞内囊泡信号的通道, #39; 源信道和 #39; 在应用掩码和 #34 之前, 在选项和 #34;D 重复通道上切换;. </li>
		<li> 在 #39; 掩码设置和 #39; 列, 选择和 #34; 常量内/外, #34; 和 #34; 将体外表面设置为: 和 #34; 将其值设置为 0.00. 
		注: 此过程将在图像中创建一个额外的通道, 在该图中, 毛细管掩码外的所有体将具有0的强度值。在毛细血管外 ( 即 , 在脑实质中) 量化事件, 选择和 #34; 将体内表面设置为: 和 #34; 并将其值设置为 0.00. </li>
		<li> 对水泡结构进行分段, 按下按钮启动和 #34; 添加新的点和 #34; 向导。使用底部箭头在 "曲面创建向导" 中的步骤之间移动. </li>
		<li> 在 "创建" 选项卡中, 使用 #34 下的下拉菜单; 源信道和 #34; 选择在步骤4.4.4 中创建的胞内掩码通道. </li>
		<li> 在 "创建" 选项卡中的 "#34"; "斑点检测和 #34;" 部分中, 将估计的 XY 直径设置为介于0.5 和1之间的值 #181; m, 根据观察到的小泡的平均大小实验。此选项确定将被分割算法检测到的最小大小. </li>
		<li> 在 "创建" 选项卡中的 "#34"、"斑点检测和 #34;" 部分中, 切换 #34 的选项; 背景减法和 #34;. 
		注意: 此选项将使用0.75 点半径的高斯滤镜 (从步骤4.4.7 中选定的值) 平滑图像, 然后减去由0.88 点半径过滤的原始图像的强度. </li>
		<li> 在 #39; 创建和 #39; 选项卡上, 按下下拉菜单, #39; 筛选类型和 #39; 选择和 #34; 质量和 #34;请注意, 这将根据点中心的强度应用阈值来分割图像。通过在 #39 上移动滑动窗口来调整较低强度阈值; 质量和 #39; 直方图直到大多数可识别的小泡被贴上标签. 
		注意: 此参数的值范围需要根据每个数据集进行经验确定. </li>
		<li> 完成 "点创建向导" 并通过按下按钮和 #34 保存创建参数; 记住参数和 #34; 在 "曲面" 菜单的 "重建" 选项卡中. </li>
		<li> 通过加载用于第一个图像的参数集来重复整个成像数据集的过程。调整和 #39 的参数; 质量强度阈值和 #39; 每个图像都要考虑背景强度的差异. </li>
	</ol> </li> </ol> 5。定量的细胞内传输在 BBB <ol> <li> 量化的音量 (在和 #181; m 3 ) 和区域 (在和 #181; m 2 ) 的毛细管段。在分析软件中, 访问步骤4.3 中创建的血管表面的统计面板。选择 #39;D etailed 和 #39; 选项卡, 然后使用下拉菜单选择和 #34; 所有值和 #34; 查找卷和区域的值. </li>
	<li> 量化毛细管段内囊泡的数量。在分析软件中, 访问在步骤4.4 中创建的点的统计面板。选择和 #39; 全局和 #39; 选项卡以查找图像中的斑点总数的值. </li>
	<li> 计算每个毛细管体积的囊泡数, 每个图像通过步骤5.1 中计算的体积来规范化斑点数。将此值乘以1000以获取每1000和 #181 的囊泡数; 毛细管体积的 m 3 . </li>
	<li> 要量化毛细管内的总强度, 可按照步骤4.3 中描述的说明创建一个覆盖整个图像的新曲面, 并进行以下修改。选择包含相关信号的通道 ( 例如 , mIgG) 作为源通道, 并将表面积详细级别的值设置为5和 #181; m。要创建覆盖整个图像的曲面, 请将较低强度阈值设置为 0. </li>
	<li> 在分析软件中, 访问在步骤5.4 中创建的曲面的 "统计" 面板。选择 #34;D etailed 和 #34; 选项卡, 然后使用下拉菜单选择和 #34; 所有值和 #34;。记录 #39 的值; 强度和 #39; 为感兴趣的渠道;此参数对应于所有像素上的单个强度值的总和. 
	注: 对于胞内信号, 这对应于复制的通道, 体外表面设置为0.0。为一个信号在脑子实质, 这对应于复制的渠道与体里面表面设置到 0.0. </li>
	<li> 计算每个毛细管体积的荧光强度, 使每个图像按照步骤5.1 中计算的体积来规范强度。将此值乘以 1000, 以获取每1000和 #181 的荧光强度值; 毛细管体积的 m 3 . 
	注意: 对于脑实质中的荧光信号, 将总的图像体积减去毛细管体积, 乘以1000以获得每1000和 #181 的总强度; m 3 的脑实质. </li>
	<li> 对整个数据集重复该过程, 并使用适当的软件对数据进行统计分析. </li>
</ol>

<ol>
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作者受雇于罗氏。

上面所述的协议描述了脑屏障的高分辨率显微镜的制备、荧光染色、图像采集和分析参数。该方法最近被用于研究抗体传递平台的定位3, 内源性 IgG 在 bbb17中的传输, 以及 bbb 在细胞损失时的异质性,18。可以修改协议中的不同步骤以适应实验的具体目标。首先, 使用厚 (100 µm) 的部分, 方便他们在免疫和安装过程中处理。它还允许3D 重建毛细管网络, 神经血管单位, 并为毛细管和 NVU 横断面的产生。然而, 抗体在组织切片中的渗透可能会有所不同, 一些抗体染色可以限制在表层的组织接近片。该协议可以通过增加洗涤剂浓度在性步骤和/或性步骤的长度, 以改善抗体渗透到组织。第二, 由于光散射以及折射率不匹配的光学像差, 图像质量可能会受到影响, 因为它在组织内部 (通常是20到30µm) 中的图像更深层。为了解决这个问题, 新的组织清除和活性抗体渗透的方法20可以与该协议相结合, 以图像更大的组织体积。再次, 在图像采集后进行反褶积, 提高图像的轴向分辨率。本协议中使用的盲反卷积算法的选择是基于 (i) 它的易用性, 因为不需要点扩展函数的计算, (ii) 它对提高图像质量的鲁棒性21, 以及 (iii) mIgG 上缺少人工制品细胞内结构的实施。根据样本中可视化的胞内结构, 其他反褶积算法可能会导致较高的图像质量。下面的参考资料21,22提供了关于图像反褶积的附加算法的优点和局限性的广泛讨论。最后, 利用图像分析软件包允许在三维度中对毛细血管和细胞内结构进行分割。显然, 图像分析不限于协议中描述的软件, 而替代包 (例如, 参考23中讨论的软件包) 可以用于分割图像。通过对图像分割精度的评估, 验证了不同软件程序对脑屏障内细胞结构分析的适用性。
由于这种方法是基于固定样本, 它不提供直接信息的动态 transcytosis 横跨 BBB。然而, 它可以与时效实验相结合, 例如, 通过静脉注射的兴趣分子和测量其在注射后不同时间点 bec 内的积累, 重建的动力学细胞内转运这种方法的优点是, 它允许对深部大脑区域进行分析, 如18所示, 活体实时成像方法目前无法访问它们。在该协议的关键步骤是仔细监测组织固定。固定与4% 粉煤灰显著降低细胞器的免疫原性和内源性或外围管理免疫球蛋白17 (图 1B)。该协议的一个限制是它的要求, 高质量的抗体 (即, 低非特异性染色, 低交叉) 适合免疫荧光。如果有这种试剂, 该方法可用于研究任何感兴趣的蛋白质的细胞内定位。例如, 该协议用于识别脑血管内皮细胞中的溶酶体17。还应该指出, 由于该协议是基于共焦显微镜, 横向分辨率受到衍射的限制, 无法解析小于大约175到 250 nm 的结构 (图 2)。
以前的研究通过共聚焦显微镜对神经血管单元的细胞组成进行了详细的分析25,26。然而, 研究 BBB 内的细胞内转运主要依赖于透射电子显微镜的使用19,27,28。虽然这种方法提供了最高的横向分辨率的细胞内结构, 电子显微镜仍然是一个具有挑战性的技术, 低吞吐量。此外, 可以被 EM 可视化的不同分子靶数是非常有限的。该协议提供了一种可访问的替代方法来调查 BBB 的细胞内转运。完整的程序, 从大脑收集到图像分析, 可以在5天到6日进行。如果有合适的抗体, 免疫荧光可以同时检测多个细胞类型/分子在同一样本。此外, 该协议还可以与超分辨率显微技术相结合, 以克服空间分辨率29中的局限性。总的来说, 上述的协议, 使量化的变化, 在细胞内定位的蛋白质利益的神经血管单位。它适用于不同的遗传或药理干扰将允许调查 BBB在体内的细胞结构和传输功能。

血脑屏障 (BBB) 是一种由星形胶质细胞、周、神经元和内皮干细胞形成的连续细胞屏障, 它将中枢神经系统 (CNS) 与血液循环1分离。对血脑屏障转运的调节在维持脑内稳态方面起着至关重要的作用, 并由大脑内皮细胞 (bec) 的特殊特性介导。bec 之间存在紧密的胞间连接和低的基底率的 transcytosis 限制细胞和 transcellular 运输的血液传播分子, 分别为2。最近, transcytosis 通路在 bec 已经被利用, 以提高治疗大分子的交付给大脑3,4。然而, transcytosis 跨 BBB 的机制尚未完全被描绘成5,6。
广泛的工作已经做了体外破译细胞和分子机制, 调节细胞内传输横跨 bec7,8,9,10, 11, 但此类系统无法重述神经血管单元 (NVU) 的复杂结构和生理学。另一方面, 研究在体内12,13提供了详细的传输速率的量化信息, 但不提供深入了解的细胞内的运输机制。因此, 调查 NVU 的细胞和细胞内的成分在体内和ex 体内仍然是非常具有挑战性的14。只有有限数量的技术可以用来分析 NVU 细胞内的亚单位结构。大多数研究使用电子显微镜, 但这种技术是有限的复杂协议所需的适当组织准备和样品处理。因此, 我们建立了一个基于高分辨率共焦显微镜的方法, 这将促进脑标本的处理, 分析和量化 NVU 细胞内的亚单位。
在这里, 我们描述了一个协议, 利用小鼠大脑自由浮动的部分, 以执行定量成像的 BBB 和 NVU 在细胞和亚型水平。我们测试和验证了一些抗体的图像和重建的 NVU 在三维度。此外, 这个协议允许成像在最大的光学衍射有限的分辨率的细胞器内的脑毛细血管。结合图像分析, 本协议可用于研究不同实验条件下的大分子在血脑屏障内的细胞内转运, 如神经的小鼠疾病模型。

<table><tbody><tr><td>Rat monoclonal antibody clone ER-MP12 against CD31/PECAM</td><td>Novus Biologicals</td><td>MCA2388</td><td>Labels Brain Endothelial Cells&lt;br/&gt; Use at dilution 1:100&lt;br/&gt; detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td></tr><tr><td>Rat monoclonal antibody against Podocalyxin</td><td>R&amp;amp;D Systems</td><td>MAB1556</td><td>Labels Lumen of capillaries&lt;br/&gt; Use at dilution 1:100&lt;br/&gt; detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td></tr><tr><td>Rabbit polyclonal antibody against CollagenIV</td><td>Biotrend</td><td>BT21-5014-70</td><td>Labels Basement membrane of capillaries&lt;br/&gt; Use at dilution 1:100&lt;br/&gt; detect with donkey polyclonal anti-rabbit IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td></tr><tr><td>Goat polyclonal antibody against CD13</td><td>R&amp;amp;D Systems</td><td>AF2335</td><td>Labels pericytes&lt;br/&gt; Use at dilution 1:100&lt;br/&gt; detect with donkey polyclonal anti-goat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td></tr><tr><td>Mouse monoclonal antibody clone G-A-5 against GFAP coupled to Cy3</td><td>Abcam</td><td>ab49874</td><td>Labels Astrocytes&lt;br/&gt; Use at dilution 1:100</td></tr><tr><td>Rabbit polyclonal antibody against Iba-1</td><td>Wako</td><td>019-19741</td><td>Labels Microglia&lt;br/&gt; Use at dilution 1:100&lt;br/&gt; detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td></tr><tr><td>Rat monoclonal antibody ABL93 gainst LAMP2</td><td>Fitzgerald</td><td>10R-CD107BBMSP</td><td>Labels Lysosomes&lt;br/&gt; Use at dilution 1:100&lt;br/&gt; detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td></tr><tr><td>donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor594</td><td>LifeTechnologies</td><td>A21203</td><td>Use at dilution 1:400</td></tr><tr><td>donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor488</td><td>LifeTechnologies</td><td>A21202</td><td>Use at dilution 1:400</td></tr><tr><td>goat polyclonal antibody against mouse IgG (H+L) AlexaFluor555</td><td>LifeTechnologies</td><td>A21422</td><td>Use at dilution 1:400</td></tr><tr><td>Filter paper</td><td>Sigma-Aldrich</td><td>WHA10334347</td><td>Whatman prepleated qualitative filter paper&lt;br/&gt; Grade 0858 1/2, grained</td></tr><tr><td>Cyanoacrylate glue</td><td>Roth Carl</td><td>258.1</td><td>Roti Coll 1</td></tr><tr><td>Fluorescent Mounting medium</td><td>Dako</td><td>S3023</td><td>Dako fluorescent mounting medium</td></tr><tr><td>Adult mice</td><td>The Jackson Laboratory</td><td>000664</td><td>C57BL/6J male or female mice&lt;br/&gt; between 9 and 19 months of age</td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>14047235612</td><td>Leica VT100S vibrating blade microtome</td></tr><tr><td>Laser Scanning Confocal microscope</td><td>Leica Microsystems</td><td>NA</td><td>Leica TCS SP8 X with HyD detectors&lt;br/&gt; and White light laser</td></tr><tr><td>Image processing software</td><td>Leica Microsystems</td><td>NA</td><td>Leica Application Suite AF version 3.1.0 build 8587</td></tr><tr><td>Image analysis software</td><td>Bitplane scientific software</td><td>NA</td><td>Imaris version 7.6.5 build 31770 for x64</td></tr><tr><td>Fluorescence SpectraViewer</td><td>ThermoFisher Scientific</td><td>NA</td><td>https://www.thermofisher.com/ch/en/home/life-science&lt;br/&gt; /cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</td></tr></tbody></table>

high-resolution confocal imaging of the blood-brain barrier: imaging, 3d reconstruction, and quantification of transcytosis

血脑屏障 (BBB) 是一种动态的多细胞界面, 调节分子在循环和大脑之间的传输。Transcytosis 横跨 BBB 调节激素, 代谢物, 和治疗性抗体的大脑实质的交付。在这里, 我们提出了一个协议, 结合免疫荧光的自由漂浮部分与激光扫描共聚焦显微镜和图像分析, 以可视化的细胞器内的细胞在血脑屏障。将此数据与3D 图像分析软件结合在一起, 可以对毛细血管体积和表面积的半自动分割和定量, 以及 BBB 内细胞器的数量和强度。用小鼠内源性免疫球蛋白 (IgG) 检测细胞内囊泡, 并在血脑屏障上进行量化。该协议可用于研究不同分子的脑血脑屏障 transcytosis 的机制, 即体内。

作为从这里所描述的协议中获得的图像的典型例子, 小鼠脑切片上的抗体被识别出 NVU 的不同成分, 包括基底膜、星形胶质细胞、周和内皮细胞膜 (见用于特定抗体的材料表(图 1A、D、和E)。在这个分辨率下, 可以区分个别的星过程和与毛细血管直接接触的端脚。
为了强调该协议对检测细胞内结构的适用性, 从周边注射了人类抗 Tau mAb86 抗体16的动物脑切片染色了荧光标记的人抗体 (图 1B)。mAb86 是已知的具体目标神经元表达的病理形式头16。使用本文所述的协议, 在神经元内的单个水泡结构内检测到 mAb86 的衍射限制分辨率 (图 1B-C)。此外, 内源性小鼠 IgG 检测在内皮细胞内部结构, 但不是在周 (图 1D-E和图 2)。
获得高分辨率的脑血管的共焦 z 栈可以在 BBB 中进行三维的毛细血管和胞内泡的分割。图 2显示了呈现和分割带有胶原和小鼠 IgG 阳性胞内泡的毛细血管标记过程的示例。通过量化一个完整的分割图像的数据集, 例如通过测量每毛细管体积泡的数量, 可以研究在不同条件下的细胞内传输过程的变化。图 3b-C显示了 mIgG 泡数和荧光强度的差异, 对应于脑实质的 mIgG, 分别在血小板生长因子-b 中的细胞损耗鼠标模型, 如先前报告的17. 同样的方法最近也被用来分析不同大脑区域之间血脑屏障内转运的变化18。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56407/56407fig1.jpg"> 
图 1:神经血管组织多细胞类型和亚单位结构的标记.具有本协议获得的神经血管单元 (A和D) 和细胞内囊泡 (B) 或内皮细胞 (E) 中的有代表性的图像。在A (顶部) 中的最大强度投影图像显示了胶原 (绿色) 周围的毛细血管内的胶质细胞阳性星状突起 (红色) 的分布。箭头指向单个星进程。缩放条 = 20 µm。在这个分辨率下, 单个星形胶质细胞的过程和端脚是清晰可见的, 如在盒装区域 (底部) 的放大图像中所示。箭头指向星端脚。缩放条 = 10 µm。在B中的图像显示了在海马神经元内 mAb86 (绿色) 的外围注射抗体的积累。箭头指向单个 mAb86-positive 泡。缩放条 = 10 µm。C中的关系图显示单个泡的线轮廓强度。囊泡大小估计从全宽的一半最大的高斯拟合 (黑实线) 的强度曲线 (绿线和圆圈)。D中的图像显示了一个三维重建的内皮细胞 (绿色), 周围的细胞 (红色) 在基底叶片 (胶原, 灰色)。下面板显示了单个荧光通道。缩放条 = 10 µm。E中的图像显示 mIgG (红色) 在内皮细胞内囊泡中的定位 (左面板, CD31 绿色), 但不周 (右面板, CD13 绿色)。在所有图像中, DAPI 染色的细胞核显示为蓝色。缩放条 = 5 µm. 已从引用17中修改了面板 D 和 E。<a href="//ecsource.jove.com/files/ftp_upload/56407/56407fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56407/56407fig2.jpg"> 
图 2: 三维毛细血管和胞内囊泡在血脑屏障上的呈现.通过所述的协议, 获得了胶原阳性毛细血管 (绿色) 和小鼠 IgG 胞内泡 (红色) 的高分辨率共焦图像 (A)。左面板显示带有剖面的单个光学截面。箭头指向脑内皮细胞内的单个 mIgG 阳性囊泡。右侧的面板显示了使用图像处理软件的完整 z 堆栈的3D 重建 (请参见材料表)。毛细管体积 (B) 和单个囊泡 (C) 以三维度呈现, 并使用图像处理软件进行量化 (参见材料表)。在所有图像中, DAPI 染色的细胞核显示为蓝色。缩放栏 = 5 µm.<a href="//ecsource.jove.com/files/ftp_upload/56407/56407fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56407/56407fig3.jpg"> 
图 3: 定量的 mIgG 细胞内定位在 BBB.具有代表性的图像显示 (A) 三维毛细血管重建 (标签为胶原、绿色) 和 C57BL/6 小鼠的 mIgG (红色) 分布, 在血小板生长因子-b 中, 细胞衰竭的小鼠以前在19中进行了描述。缩放条形图 = 10 µm。B中的图像显示了胶原掩码内的胞浆内部泡的分割。届B中的 e 图显示了每个毛细血管体积的 mIgG 泡数的量化和比较。每个点表示来自单个毛细管段的测量。实线显示平均值, 误差条表示数据的标准偏差。C中的图像在胶原掩码外显示 mIgG 荧光信号. 同样, 在C中的图显示了 C57BL/6 小鼠和血小板生长因子-b 细胞衰竭小鼠的脑实质中 mIgG 荧光强度的定量和比较。荧光强度单位是由所有 C57BL/6 小鼠的平均 mIgG 软组织强度正常化。此图已从引用17中进行了修改。<a href="//ecsource.jove.com/files/ftp_upload/56407/56407fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis at JoVE.com

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

在这里, 我们提出了一个 microscopy-based 协议的高分辨率成像和三维重建的小鼠神经血管单位和血脑屏障使用脑漂浮部分。这种方法可用于血脑屏障内细胞器的可视化、分析和量化。

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

high-resolution-confocal-imaging-blood-brain-barrier-imaging-3d

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.7K 次观看.  Roche Innovation Center Basel. 在这里, 我们提出了一个 microscopy-based 协议的高分辨率成像和三维重建的小鼠神经血管单位和血脑屏障使用脑漂浮部分。这种方法可用于血脑屏障内细胞器的可视化、分析和量化。

视频: High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

通过颅窗口在新皮层的血流量为双光子成像方法

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

科研

生物学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

神经科学

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

癌症研究

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

使用有机体片 3D 模型、机器学习和共声断层扫描量化脑转移肿瘤微环境

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

成像在细胞水平上通过3DISCO清除完整生物系统

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

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

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

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 ...