提交人希望承认由 Leduq 基金会资助的 Sphingonet 财团支持这项工作。这项工作还得到了合作研究中心 "血管分化和重塑" (CRC/Transregio23, 项目 C1) 和7的支持。FP, COFUND, 歌德国际博士后方案进入, 291776 号资金。我们进一步承认凯瑟琳索默她的技术援助与老鼠的处理和基因分型。

在手术过程中, 所有动物都非常小心的处理, 尽量减少疼痛或不适。本程序遵循本机构的动物保育准则, 并经当地委员会批准 (Regierungspraesidium 达姆施塔特, 批准号 FK/1044)。
图 1显示了鼠标中体内渗透性检测的工作步骤示意图。每个步骤的详细信息如下所述。
1. 动物搬运
<ol><li>
示踪剂和麻醉剂的制备与管理
	<ol>
<li>在渗透率测定前至少一天为组织嵌入的标记管和模具准备。在清洁通风罩下工作, 使用80% 乙醇清洗的工具, 在整个协议中保持无菌状态。使用雄性或雌性野生型 (Ang-2) 或 angiopoietin-2 (GOF) CD1 小鼠在成熟的成年年龄组3-6 月为当前协议。按照前面描述的10对小鼠进行基因分型。 注: 80% 酒精不会导致无菌器械, 但会减少所制备样品的污染。</li>
		<li>将无菌 PBS 中的所有示踪剂稀释成2毫米的股票, 并将保护的整除数保存在-20 摄氏度。</li>
		<li>选择示踪剂, 如 (四甲基罗丹明) 和 (异硫氰酸荧光素) FITC 具有不同的激发/发射光谱, 是赖氨酸可以修复, 导致在染料之间的最小干扰, 荧光显微镜 (使用FITC 过滤器) 和通过荧光法在平板阅读器中进行渗透率测定。 注: 葡聚糖 3 kd 和 FITC 葡聚糖 3 kd 在研究中都是赖氨酸可修复的, 因此也可以用于固定化醛后的免疫组化分析, 以调查区域差异。</li>
		<li>遵循机构护理指南, 尽最大的努力处理所有动物。在1:1 混合10、13中使用每个跟踪器的100µL 组合时, 用100µL 示踪解决方案注入腹腔每个鼠标, 可增加多达200µL。注射至少一个动物与 PBS 单独而不是示踪剂, 作为假的控制自体荧光背景减法。</li>
		<li>5分钟后示踪剂注射, 麻醉动物与 i. p 注射液氯胺酮和甲苯噻嗪 (100 毫克和5-10 毫克在0.9% 生理盐水每公斤体重分别, 150 µL 的鸡尾酒每25克鼠标)。 注: 在手术过程中, 由于动物麻醉和灌注/牺牲之间的时间短暂 (10 分钟), 没有观察到任何干燥的眼睛, 兽医软膏没有在眼睛上应用。</li>
	</ol>
</li>
	<li>
血液采集与心脏灌注
	<ol>
<li>麻醉后为心脏灌注10分钟准备动物。检查没有爪子抽搐反应, 以确保动物达到手术平面的麻醉。</li>
		<li>将动物放在背上, 在腹部皮肤上涂抹80% 乙醇。使用小剪刀, 打开腹壁与小切口 (2 厘米) 刚刚下方的肋骨笼。分开肝脏从膜片然后慢慢地切开通过膜片暴露胸膜腔15。</li>
		<li>将肋骨保持在双边和修复切口胸骨, 以暴露左心室。在左心室后部插入一个21尺的蝶形针, 连接到蠕动灌注系统。</li>
		<li>穿刺右心房, 并迅速 (在十年代) 收集200-300 µL 血液释放到胸腔使用1毫升吸管提示 (末端切断) 在血清收集管和储存在冰上。 注: 这种灌注过程是不生存的。</li>
		<li>一旦血液收集, 打开灌注系统 (10-12 rpm, 5 毫升/分钟) 和灌注动物为3分钟与温暖 (RT) 1x PBS (免费的 Ca2 +/毫克2 +离子)。 注意: 在灌注过程中, 含有 Ca2 +/毫克2 +离子的 PBS 可以用于更好的心脏活动。用于灌注的 PBS 总数量在15-20 毫升范围内。</li>
		<li>通过注意肝脏、肾脏的颜色来评估灌注质量, 在灌注后呈白色/苍白。 注: 肾脏或肝脏灌注不一定表明脑灌注, 因为动脉 (颈动脉/椎体) 到达大脑从主动脉可能会在3的准备过程中, 特别是在心房穿刺期间破裂。</li>
	</ol>
</li>
</ol>2. 组织加工
<ol><li>器官收集和存贮
	<ol>
<li>在灌注结束时, 通过颈椎脱位确认动物死亡, 并获得大脑和肾脏。通过大脑的颜色来验证脑灌注 (脑膜血管中没有可见的血液), 以便在灌注质量不好时排除动物的渗透率分析。使用手术刀将大脑分成 2 hemibrains (图 1)。</li>
		<li>解剖与手术刀一 hemibrain 没有嗅觉裂片和小脑和转移 hemicerebrum 到一个2毫升管。如果需要小脑通透性评估, 则将半小脑储存在额外的导管中。</li>
		<li>把一个肾脏转移到另一个2毫升的试管里。立即将样品存放在干冰上。</li>
		<li>本机嵌入在干冰上的最佳切削温度 (o.c. T) 化合物的剩余肾脏和半脑 (包括小脑和嗅觉裂片)。</li>
		<li>最后, 离心机在 10, 000 克, 10 分钟4°c 的血液标本储存在冰上。将血清上清液转移到1.5 毫升管并放置在干冰容器中。</li>
		<li>将收集到的所有样品在干冰上转移到-80 °c 冷藏库, 直到进一步加工。 注: 在进行均匀化步骤之前, 冷冻样品是很重要的, 因为在冷冻解冻后均匀化效率增加。</li>
	</ol></li>
	<li>均匀化和离心
	<ol>
<li>冰上解冻的半脑和肾脏样本冻结在-80 摄氏度, 并称管含有器官。从这些权重, 减去几个空管的平均值 (约 20), 以获得组织重量。将300µL 和200µL 的冷 1X PBS 分别添加到含有肾脏和半大脑的管中。 注: 对于低剂量的组织 (如半小脑, 低于100毫克) 体重的个别管的建议。</li>
		<li>融汇在原来的离心管中的每个样品与一个聚四氟乙烯 (PTFE) 杵连接到一个电动顶部搅拌器 (1, 000 rpm) 通过执行约15冲程 (1 冲程 = 1 和1下来)。在样品之间用 PBS 冲洗杵, 然后擦干, 然后再进行下一个样品。 注意: 在均匀化过程中使用洗涤剂是避免的, 因为可能会干扰荧光法。然而, 在本协议中也检测到细胞内示踪物, 因为组织完全均匀化, 在一轮冰冻解冻后效率更高。</li>
		<li>将均匀的样品保存在光和离心机的冰上, 最后在1.5万克, 20 分钟, 4 摄氏度的桌面离心机上。将上清液转移到新的1.5 毫升的冰上, 以便立即进行荧光或-80 °c 的分析。</li>
	</ol></li>
	<li>荧光测量与定量
	<ol>
<li>如果在上一步结束时冻结, 在冰上解冻的组织上清和血清标本储存在-80 ˚C 保护他们从光与箔。</li>
		<li>吸管50µL 稀释血清 (30 µL 1x PBS + 20 µL 血清) 或组织上清液 (as) 成384井黑板。包括至少一个来自假动物的血清和组织上清液的样本, 以确保适当的背景减法, 因为这种组织匀浆背景通常高于作为稀释剂的 PBS。 注: 平均3只假动物可用于自体荧光, 尽管在假的自发荧光值 (未显示的数据) 中有很小的变异性。用 PBS 稀释后可降低血清用量, 也可提高低荧光样本 (如脑样) 的信噪比。对10µL 血清进行了测试, 稀释40µL PBS。确保井里没有气泡。</li>
		<li>将384井黑板插入板读取器, 并打开一个新的脚本选择荧光测量。</li>
		<li>将增益设置为最佳, 并分别使用550/580 或490/520 的激发/发射 (nm) 值 (FITC dyee), 并开始测量以获得原始荧光单元 (RFUs)。</li>
		<li>使用原始荧光单元 (RFUs) 从板读取器计算渗透率指数 (PI) 后减去相应的假值。<ol>
<li>
* 渗透率索引(毫升/克)= (组织 RFUs/g 组织重量)/(血清 RFUs/毫升血清)
</li>
		</ol>
</li>
		<li>动物 GOF1 的脑通透指数 (PI) 的示例计算 (表 1):<ol>
<li>GOF1 脑 RFUs-假脑 RFUs = 154-22.5 = 131。5</li>
			<li>GOF1 血清 RFUs-假血清 RFUs = 38305-27 = 38278</li>
			<li>脑重量 (g) = 0.195</li>
			<li>血清容积 (毫升) = 0.02</li>
			<li>脑通透性指数 (10-3毫升/克) = (131.5/0.195)/(38278/0.02) = 0.352 注: 渗透率指数计算产生的价值是绝对的和可比性的实验和组织类型。然而这些可以被提出作为比率为容易比较在2个小组之间12。这可以通过将两组动物的 pi 与对照组的平均 pi 进行分割, 从而使对照组的平均值达到 1, 同时保持对照组的动物间变异。此转换为相对于控件组设置为1的实验组 (或组) 提供值。</li>
		</ol>
</li>
	</ol></li>
	<li>示踪剂的免疫荧光可视化
	<ol>
<li>将本机内嵌入的肾脏/半脑块 (o.c. T) (步骤 2.1) 切成10µm 节, 恒温器设置为-20 摄氏度, 并将切片转移到放置在室温下的幻灯片。一旦部分被烘干 (大约30分钟), 转移幻灯片到-80 °c 冷冻机, 直到使用。</li>
		<li>染色当日, 将37摄氏度的滑梯解冻10分钟, 然后在室温下用 4% paraformaladehyde (粉煤灰) 固定部分, 然后在 PBS 中快速清洗。</li>
		<li>在室温下, 用含有 1% BSA 的无菌 PBS 和0.5% 海卫 X-100 pH 7.5 为1小时的通透/阻塞缓冲区孵化部分。</li>
		<li>用 CD31 原抗体 (0.5 毫克/毫升, 克隆肌 13.3) 在室温下孵化1.5 小时的幻灯片, 在1:100 的缓冲中稀释, 含无菌 PBS, 0.5% BSA, 0.25% 海卫 X-100 (pH 7.2), 随后三5分钟清洗 PBS。</li>
		<li>在上述缓冲区中进行二次抗体孵化, 在室温下使用特定物种的荧光标记抗体稀释 1:500 (2 毫克/毫升)。对于 CD31, 山羊抗鼠 alexa 568 或 alexa 488 可以使用稀释1:500。包括 DAPI (300 µM) 的二次抗体组合 (1:1, 000 稀释从股票) 染色的细胞核。</li>
		<li>将染色的部分装上水 polymount, 在天黑时将其留在室温聚合。</li>
		<li>利用光谱成像共聚焦激光扫描显微镜系统获取图像。通过 NIS 元素软件分析图像 (版本 4.3)。软件, 如 Photoshop 可以用于生成蒙太奇数字。</li>
	</ol></li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier:+Role+of+Brain+Endothelial+Surface+Charge+and+Glycocalyx.">Blood-Brain Barrier: Role of Brain Endothelial Surface Charge and Glycocalyx.</a> Ischemic Blood Flow in the Brain. , 33-38 (2001).</li><li>Fu, B. M., Li, G., Yuan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+effects+of+the+blood-brain+barrier+on+the+transport+of+charged+molecules.">Charge effects of the blood-brain barrier on the transport of charged molecules.</a> The FASEB Journal. 22 (1 Supplement), (2008).</li><li>Goebl, N. A., Babbey, C. M., Datta-Mannan, A., Witcher, D. R., Wroblewski, V. J., Dunn, K. 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作者声明他们没有竞争的财政利益。

血脑屏障功能障碍与一系列神经系统疾病有关, 包括原发性和次级脑肿瘤或中风。脑屏障破裂通常与危及生命的中枢神经系统水肿有关。因此, 分子机制的阐明, 触发开放或关闭的脑屏障是治疗的意义, 在神经系统疾病和一般调查的研究人员。然而, 研究文献中报告的脑屏障通透性在体内的方法往往与技术上的困难有关, 这取决于荧光图像的单调和主观量化,17,18,19. 此外, 有些方法包括全脑荧光成像, 这是谬误的, 因为它更表明了表面的血管通透性不构成一个严密的屏障。即使根据客观的脑组织吸收 (如埃文斯蓝渗透率评估) 进行量化, 这些动物通常不会因为大量的血流而导致错误的解释。脑重量是另一个经常不包括在渗透率计算中的变量, 但需要作为血管通透性引起的水肿可以增加重量和改变净通透性。此外, 性和动物对动物的大脑重量变化可以云渗透率差异。放射性示踪剂, 如14C 蔗糖和 [3H] 菊粉已成功地应用于脑通透性指数, 但不提供范围广泛的大小和电荷的渗透率调查, 与荧光标记示踪剂20。
基于上述原因, 我们采用了一种简单、客观和定量的方法, 用荧光标记示踪剂估计小鼠血脑屏障通透性体内。Dextrans 是以中、高分子量 (3-200 kD) 为特征的亲水性多糖, 可根据共轭荧光获得带电或带电分子。内皮紧密连接由主要 claudins (1, 3, 5 和 12) 和 occludin 组成, 共同确定旁细胞大小和电荷选择性由存在于其第一胞外循环 (在21中复查) 的电荷孔隙残留物.跨血脑屏障的渗透性在体内也依赖于糖萼和基底叶片, 两个结构存在于 BBB 22, 23 的两边.Luminally 目前糖萼是一种由负电荷寡糖 (肝素硫酸盐) 形成的凝胶状结构, 也可作为血液传播大分子 (如白蛋白) 的屏障。糖萼厚度或成分的变化也与血管通透性的变化有关, 正如我们观察到的 angiopoietin-2 增益的功能小鼠相比, 野生型小鼠 10.而基底膜则存在于内皮细胞的 abluminal 侧, 由内皮细胞和毛细血管构成的血管基底膜组成, 而实质基底膜或胶质基底膜则由由星形胶质细胞。这些基底膜也有负电荷, 因此也有能力充当电荷屏障。在这方面, 共轭 dextrans 提供了大小和电荷基渗透率的调查。例如, 葡聚糖 3 kd 为阴离子, 而 TXR-葡聚糖 3 kd 是中性的, 结合其中一种示踪剂与共轭高分子量葡聚糖, 如 FITC 葡聚糖 70 kD 在同一混合注射到小鼠, 可以评估的大小为基础的渗透性。进一步利用荧光标记 dextrans 的赖氨酸可修复性, 探讨标准免疫荧光染色中渗透率的区域差异。Dextrans 通常也表现出低免疫原性, 因而作为良好的示踪剂。路西法黄色, 尸胺, fluorescein-5-thiosemicarbazide (FTSC), 是在低分子量范围 (0.4-0.8 kD) 的其他示踪物和是能被修复的。
在我们的方法中, 用 i. p 路线注射示踪剂, 灌注小鼠, 获得脑组织匀浆的荧光, 对湿重和血清荧光进行规范化。这是一个简单而高度定量和客观的方法, 因为没有选择的剖面/图像, 在量化的 immunofluorecence 方法, 是经典的使用。我们只利用一个半脑的渗透性检测, 并利用其他 hemibrain 免疫荧光染色通过分析的矢状切片提供区域差异的同一动物。使用每个半脑为不同的并行测量是我们的协议的主要优势之一。此外, 其他器官如肾脏、肝脏等的通透性作为良好的内部控制, 因为这些器官的基底渗透性很高。为了比较2个小组, 你必须首先减去假动物 (没有接受示踪剂注射) 从所有动物在两个小组和所有组织类型 (肾脏, 脑子和血清) 的荧光从每个追踪器, 然后继续规范化对2组进行比较的计算。以组织与血清荧光的比值为组织重量和血清体积, 提出了渗透率指数 (PI)。我们的计算结果对示踪剂渗透率具有绝对价值, 可以比较实验和组织类型。但是, 这些值可以很容易地表示为比我们之前12所做的2组之间的比率或百分比。这可以通过将每种动物的 pi 除以对照组中所有动物的平均 pi 来实现。这将驱动控制组 PI 通过 1, 但保持在动物之间的错误和为实验组提供的价值, 相对于控制组的平均值为1。
用 i. p 注射示踪剂比较容易, 但与 i. v 注射液相比, 成本更高, 因为追踪器的用量需要增加。我们的数据 (未显示) 在这方面表明, 在示踪剂中, 几乎有2倍高的量是我所需要的. p 注射液与 i. 型相比, 可获得类似的血清荧光值。同时, 由于追踪器吸收到血流中的时间, 与 i. v 相比, 环流的时间更高。在这方面, i. p 注射液的血清荧光值为15分钟后, 在低、中分子量范围内的示踪剂中, 0.4-4 kD (未显示数据) 可媲美5分钟. v 注射液。我们建议短暂的循环时间, 因为旁细胞通透性是线性的, 如果有相当多的组织荧光检测后灌注后短暂的循环时间 (15 分钟), 它已经表明了通透性表型, 因为我们已经报告在我们的以前的出版物10,12,13,14。但同时可以获得血清荧光的规范化。在较长的循环时间, 组织间隙是相当可观的, 这大大降低了血清荧光, 从而可能影响渗透率的价值正常化的血清。当与追踪器的大小/电荷有关的循环时间必须对每个场景进行优化时, 我们建议以短循环时间为出发点。这也适用于更高的分子量溶质, 如血浆 IgGs 和纤维蛋白原 (150-400 kD), 其渗透特性不同于惰性葡聚糖示踪物, 由于其极大的大小和特定的相互作用与细胞受体, 如 Fc改变其半衰期24的受体。另一方面, Dextrans 在内皮层25上没有任何特定的传输系统, 由于膜泡相关蛋白 (PLVAP) 26 的水平非常低, 脑内皮细胞不经历吞饮显著的水平., dextrans 的主要运输路线是旁细胞。在转基因小鼠的渗透性与野生型窝相比, 我们成功地应用了上述方法, 并在治疗干预后对野生型小鼠的渗透性变化进行了评估10,12,13,14. 总之, 结合免疫荧光染色, 这里描述的通透性检测是一个简单的和稳健的方法来评估小鼠的渗透性的在体内, 可以也适用于其他小动物。

血脑屏障 (BBB) 由紧密相关的毛细血管 (pc) 支持的微血管内皮细胞 (ECs) 组成, 它们是基底叶片中的 ensheathed, 而星形胶质细胞 (ACs) 将基底膜包裹在其端脚上1 ,2。ECs 与支持和调节屏障功能的几种细胞类型相互作用, 主要是 ACs 和 pc, 以及神经元和小胶质, 它们共同构成神经血管单元 (NVU)。NVU 对脑屏障功能至关重要, 它限制了血液传播毒素和病原体进入大脑的传输。这一功能是紧密连接分子, 如 claudin-5, occludin, 拼装 occludens-1, 这是存在之间的, 也由于转运体, 如 p-糖蛋白 (p gp) 的作用, 外流分子进入内皮回到容器流明1,2,3。然而, 在 EC 等离子膜1,2,3上表达的特定运输者, 血脑屏障允许运输必要的分子, 如营养素 (葡萄糖, 铁, 氨基酸)。欧共体层是高度极化的在不同的运输载体之间的分布 (血面) 和 abluminal (脑面膜), 以允许特定和向量传输函数4,5.虽然脑屏障在严密调节中枢神经系统环境方面是有保护作用的, 但对于中枢神经系统药物在帕金森氏症和功能性脑屏障等疾病中的传递来说, 这是一个重大挑战。即使在神经疾病的脑屏障功能障碍, 它不能假设, 大脑药物传递是增加特别是因为屏障功能障碍可能包括损害特定的运输目标, 例如阿尔茨海默病 (AD)。在 AD 中, 一些淀粉样β转运体, 如 LRP1, 愤怒, P gp 被称为失调, 因此针对这些转运可能是徒劳的6,7,8。脑屏障损伤在一些神经疾病, 如中风, AD, 脑膜炎, 多发性硬化症, 并在脑瘤9,10,11。恢复屏障功能是治疗策略的重要组成部分, 因此其评估是至关重要的。
在这项工作中, 我们已经描述了一个目标和定量协议的渗透率检测啮齿目动物, 我们成功地应用到几个鼠标线的转基因和实验性疾病模型10,12,13 ,14。该方法是建立在简单的腹腔注射荧光示踪剂的基础上, 然后通过灌注小鼠来去除血管腔内的示踪物。脑和其他器官被收集后灌注和渗透率评估的客观和绝对渗透指数的基础上的荧光测量组织组织匀浆在一个板块阅读器。所有原始的荧光值都是用组织组织匀浆或不接受任何示踪的假动物的血清来矫正的。大量的标准化包括在血清体积, 血清荧光和组织的重量, 从而产生的通透性指数是绝对的和可比的实验和组织类型。为了便于组之间的比较, 绝对渗透率索引值可以很容易地转换为比我们以前执行的12。同时, 储存的半脑和肾脏可用于示踪剂可视化的荧光显微术10。经典荧光显微术对获得区域通透性的差异有很高的价值, 尽管由于对组织切片和图像的主观选择进行了半定量的分析。详细步骤载于协议中, 并酌情添加注释。这为在小鼠中成功执行体内通透性检测提供了必要的信息, 可以将其扩展到其他小动物身上。该方法可应用于多种示踪剂, 通过与不同荧光光谱的示踪物相结合进行电荷和尺寸的渗透率评估。

<table>
	<tbody>
		<tr>
			<td>Tetramethyl Rhodamine (TMR) dextran 3kD</td>
			<td>Thermosfisher</td>
			<td>D3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC) dextran 3kD</td>
			<td>Thermosfisher</td>
			<td>D3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Ketavet)</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Saline</td>
			<td>Fresenius Kabi Deutschland GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldhehyde solution</td>
			<td>Sigma</td>
			<td>252549-1L</td>
			<td>prepare a 4% solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, fraction V</td>
			<td>Roth</td>
			<td>8076.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>rat anti CD31 antibody, clone MEC 13.3</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rat alexa 568</td>
			<td>Molecular Probes</td>
			<td>A-11077</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rat alexa 488</td>
			<td>Molecular Probes</td>
			<td>A-11006</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua polymount</td>
			<td>Polyscience Inc</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge butterfly needle</td>
			<td>BD</td>
			<td>387455</td>
			<td></td>
		</tr>
		<tr>
			<td>serum collection tube</td>
			<td>Sarstedt</td>
			<td>41.1500.005</td>
			<td></td>
		</tr>
		<tr>
			<td>2mL eppendorf tubes</td>
			<td>Sarstedt</td>
			<td>72.695.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech precision wipes tissue wipers</td>
			<td>Kimberley-Clark Professional</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well black plate</td>
			<td>Greiner</td>
			<td>781086</td>
			<td></td>
		</tr>
		<tr>
			<td>slides superfrost plus</td>
			<td>Thermoscientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE pestle</td>
			<td>Wheaton</td>
			<td>358029</td>
			<td></td>
		</tr>
		<tr>
			<td>electric overhead stirrer</td>
			<td>VWR</td>
			<td>VWR VOS 14</td>
			<td></td>
		</tr>
		<tr>
			<td>plate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microm GmbH</td>
			<td>HM 550</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon C1 Spectral Imaging confocal Laser Scanning Microscope System</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic perfusion system</td>
			<td>BVK Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo blood-brain barrier permeability assay in mice using fluorescently labeled tracers

血脑屏障 (BBB) 是一种专门的屏障, 保护大脑微环境的毒素和病原体在循环和保持大脑稳态。屏障的主要部位是脑毛细血管内皮细胞, 其屏障功能来自于细胞膜上表达的紧密细胞间结和外排转运体。这个功能是由毛细血管和星形胶质细胞组成的神经血管单元 (NVU) 调节。一些神经系统疾病, 如中风, 阿尔茨海默病 (AD), 脑肿瘤与受损的 BBB 功能。因此, 评估脑屏障通透性对于评估神经系统疾病的严重性和所采用的治疗策略的成功至关重要。
我们提出了一个简单而稳健的渗透试验, 已成功地应用于几个小鼠模型, 遗传和实验。该方法与常用的示踪荧光分析法相比, 具有较高的定量和客观性。在这种方法中, 小鼠被注射腹腔的混合水惰性荧光示踪剂后麻醉小鼠。动物的心脏灌注是在收割大脑、肾脏或其他器官之前进行的。器官是均匀的和离心的, 其次是荧光测量从上清。在灌注前从心脏穿刺中提取的血液为规范化目的的血管室。组织荧光对湿重和血清荧光进行规范化, 获得定量示踪剂通透性指数。为进一步确认, 对侧半脑保存的免疫组化可用于示踪荧光显示的目的。

我们最近表明, angiopoietin-2 (Ang-2) 增益功能 (GOF) 小鼠脑血管通透性比控制小鼠在健康条件下10。在脑卒中诱导的小鼠中, GOF 小鼠的梗死面积较大, 通透性大于对照窝。这些结果显示 Ang-2 在脑屏障通透性中的重要作用。因此, 该议定书利用 GOF 小鼠, 并将其与对照窝来描述体内通透性试验。然而, 这种方法可以应用于任何疾病模型, 转基因小鼠模型或药物治疗, 改变的脑屏障通透性, 因为我们以前的10,11,12,13。
有一个短的循环时间 (15 分钟) 的渗透率分析建议, 因为更长的循环时间将导致更大的清除从血管室, 这已经在以前的研究中也观察到16。清除 3 kD FITC-葡聚糖在 2 h (图 2 C, D)大于在15分钟(图 2 A, B)在肾脏并且在脑组织。在大脑中, 在这些成年小鼠 (图 B, D) 中, 由于完整的血脑屏障, 几乎没有外示踪剂。应用该方法对 Ang-2 GOF 与窝的示踪剂渗透率进行了比较。以表格格式 (表 1) 显示的结果表明, GOF 小鼠的大脑中示踪物积累量比小鼠大。然而, 肾脏荧光在这些组之间没有改变。免疫荧光图像证实了外示踪剂在 GOF 小鼠中的增加(图 3 B, D)与小鼠(图 3 A, C)相比, 追踪器仅限于血管室。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57038/57038fig1.jpg"> 图 1.使用荧光示踪剂的体内渗透性检测工作流的示意图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57038/57038fig1large.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/57038/57038fig2.jpg"> 图2。在短而长的循环时间追踪器清除.为确定渗透率测定的示踪循环时间, 将 15 min (a、B)的短循环时间与 2 h (C、D)后示踪剂注射液的长循环时间进行比较。2小时的循环时间越长, 导致肾脏中示踪物积累量很低(C) , 其中基底通透性高与短15分钟循环时间(a)可能由于非常高的清除 FITC dextran-3 kD (绿色通道) 从血管室。这种效果在大脑中更具戏剧性, 其特点是致密的血脑屏障(B, D)。CD31 染色 (红色通道) 证实了在该地区的船只存在的利益。从单一动物的代表性图像2野生型成年 CD1 小鼠注射的示踪剂为每个时间点和动物被牺牲, 没有灌注他们想象的血管内示踪物。刻度条 = 20 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57038/57038fig2large.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/57038/57038fig3.jpg"> 图3。荧光染色示踪剂评估 GOF 小鼠脑通透性的变化.在 Ang-2 GOF 小鼠(B, D)中, 相对于皮层区域中的窝(A、C) , 可可视化 3 kD-葡聚糖示踪剂 (红色) 的通透性增强。在野生动植物中, 示踪剂仅限于血管 (A、C), 而外示踪剂可在 GOF 小鼠中观察到 ( B、D中的白色箭头)。对合并图像中的 CD31 (蓝色) 进行染色(A D)确认有船只存在。图中显示2重量和 2 GOF 小鼠注射示踪剂 i. p 和牺牲15分钟后注射与灌注的代表性图像。缩放条 = 20 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57038/57038fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">动物 ID</td>
			<td rowspan="2">脑重量 (g)</td>
			<td rowspan="2">肾脏重量 (g)</td>
			<td rowspan="2">血清容积 (毫升)</td>
			<td rowspan="2">血清 RFU</td>
			<td rowspan="2">肾脏 RFU</td>
			<td rowspan="2">脑 RFU</td>
			<td colspan="2">烫发.指数 (10 ^-3 毫升/克)</td>
		</tr>
<tr>
<td>肾</td>
			<td>大脑</td>
		</tr>
<tr>
<td>GOF 1</td>
			<td>0.195</td>
			<td>0.252</td>
			<td>0.02</td>
			<td>38305</td>
			<td>31051</td>
			<td>154</td>
			<td>64。4</td>
			<td>0.352</td>
		</tr>
<tr>
<td>GOF 2</td>
			<td>0.177</td>
			<td>0.249</td>
			<td>0.02</td>
			<td>42001</td>
			<td>31411</td>
			<td>126</td>
			<td>60</td>
			<td>0.278</td>
		</tr>
<tr>
<td>1</td>
			<td>0.167</td>
			<td>0.301</td>
			<td>0.02</td>
			<td>40904</td>
			<td>31591</td>
			<td>64</td>
			<td>51。2</td>
			<td>0.122</td>
		</tr>
<tr>
<td>2</td>
			<td>0.146</td>
			<td>0.294</td>
			<td>0.02</td>
			<td>39502</td>
			<td>31768</td>
			<td>70</td>
			<td>54。8</td>
			<td>0.164</td>
		</tr>
<tr>
<td>假</td>
			<td>0.155</td>
			<td>0.27</td>
			<td>0.02</td>
			<td>27</td>
			<td>36</td>
			<td>22。5</td>
			<td>na</td>
			<td>na</td>
		</tr>
</tbody></table>表1。组织渗透性计算.Ang-2 增益函数 (GOF) 和野窝 (小鼠) 的渗透率指数计算明显显示, 与小鼠相比, GOF 小鼠的脑通透性更大 (约2倍)。然而肾脏渗透性在同一个范围在2个小组之间。由于肾脏 fenestrated 内皮不形成致密屏障, 基底肾通透性要大得多。

Watch this Scientific Journal Video about An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers at JoVE.com

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

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

在这里, 我们提出了一个小鼠脑血管通透性检测, 采用腹腔注射荧光示踪剂后灌注, 适用于动物模型的血脑屏障功能障碍。一个半脑用于定量评估渗透性, 另一个用于示踪器可视化/染色。10只老鼠的手术过程需要 5-6 小时。

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

an-vivo-blood-brain-barrier-permeability-assay-mice-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

24.2K 次观看.  Goethe University Hospital. 在这里, 我们提出了一个小鼠脑血管通透性检测, 采用腹腔注射荧光示踪剂后灌注, 适用于动物模型的血脑屏障功能障碍。一个半脑用于定量评估渗透性, 另一个用于示踪器可视化/染色。10只老鼠的手术过程需要 5-6 小时。

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

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

科研

神经科学

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 ...

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

的斑马鱼胚胎神经上皮通透性的测定

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

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 ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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 ...