Name: an in vivo blood-brain barrier permeability assay in mice using fluorescently labeled tracers
Uploaded: 2018-02-26T15:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers at JoVE.com

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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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+blood-brain+barrier+disruption:+roles+of+cyclooxygenase,+oxidative+stress,+neuroinflammation,+and+elements+of+the+neurovascular+unit.">Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit.</a> Journal of Neuroinflammation. 12, 223 (2015).</li><li>Krause, G., Winkler, L., Mueller, S. L., Haseloff, R. F., Piontek, J., Blasig, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+claudins.">Structure and function of claudins.</a> Biochimica et biophysica acta. 1778 (3), 631-645 (2008).</li><li>Johansson, B. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Fc+receptor+mediates+internalization+of+Fc+in+transfected+human+endothelial+cells.">Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells.</a> Molecular biology of the cell. 19 (12), 5490-5505 (2008).</li><li>Lopez-Quintero, S. V., Ji, X. -. Y., Antonetti, D. A., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-pore+model+describes+transport+properties+of+bovine+retinal+endothelial+cells+in+normal+and+elevated+glucose.">A three-pore model describes transport properties of bovine retinal endothelial cells in normal and elevated glucose.</a> Investigative ophthalmology &amp; visual science. 52 (2), 1171-1180 (2011).</li><li>Hallmann, R., Mayer, D. N., Berg, E. L., Broermann, R., Butcher, E. 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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,<sup class...

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?...

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

Research

JoVE Journal

Neuroscience

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

24.4K 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

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 ...

科研

癌症研究

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

医学

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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 ...

神经科学

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

免疫与感染

<meta charset="utf8"></head><body>基于荧光染料的小鼠血脑屏障分解可视化

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

<meta charset="utf8"></head><body>轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

体内 小鼠下颌下腺血管通透性检测

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

生物学

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

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利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

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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

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

体内小鼠下颌下腺血管通透性检测

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

摘要

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利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

一个

设置了一个

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

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

体内 小鼠下颌下腺血管通透性检测

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

体内小鼠下颌下腺血管通透性检测