Name: assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease
Uploaded: 2017-11-08T14:00:00.000+00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

这项工作得到了 "基金会 Neuromed" 的支持, 由意大利卫生部 "74年2月 Corrente" 资助科诺诺夫

所有动物程序均由 IRCCS Neuromed 动物保育审查委员会和 #34 批准; 依 Superiore Sanit 和 #224; #34; (许可证编号: 1163/2015-PR), 并按照欧盟指令2010的指导方针/63/欧洲动物实验. 
 1. 注射到颈静脉内的 FITC-白蛋白溶液的制备 注意: 所有实验都是在清单 (11 周龄) HD R6/2 小鼠和年龄和性别匹配的野生型 (重量) 控制中进行的。窝. 
<ol> <li> 在磷酸缓冲盐水 (PBS) 中溶解 FITC-白蛋白粉末 (参见 材料表 ), 在10毫克/毫升。
	注: 为获得最佳效果, 应为每个标签反应准备新的. </li>
</ol> 2。外科手术中使用的设备的准备工作 <ol> <li> 放置以前的蒸压的外科仪器 (请参见 材料表 )。
	<li> 用酒精 (70% 乙醇溶液) 清洗手术台。准备手术区 (45 厘米 x 45 厘米) 与外科无菌一次性毛巾悬垂和设置操作显微镜. </li>
</ol> 3。FITC-白蛋白输注到颈静脉的手术方法 <ol> <li> 记录麻醉时的鼠标体重. </li>
	<li> 麻醉鼠腹腔注射 (i. p) 的适当剂量氯胺酮 (100 毫克/千克)-嗪 (10 毫克/千克) 溶液. </li>
	<li> 在 Walantus et al. 中, 通过轻柔的脚趾掐缩反应来监测动物镇静反应, 如下所示 11 , 12 . </li> <li> 将麻醉鼠标置于正面仰卧位置, 并用胶带固定在手术台上的四条腿和尾部. </li>
	<li> 在鼠标安全后, 小心地用电动剃须刀将颈部适当的手术边缘剃掉 (参见 材料表 ), 并用聚维酮碘溶液和70% 乙醇对其进行消毒. </li>
	<li> 用棉签将裸露的剃光表面烘干. </li>
	<li> 轻轻地拉起剃光的皮肤, 用手术刀做一个1.5 厘米长的纵向切口, 在锁骨线上, 从胸部的中段开始, 延伸到颈部以下. </li>
	<li> 在显微镜观察 (5X 放大倍率) 下, 仔细地将结缔组织与镊子分开, 以暴露颈外静脉. </li>
	<li> 确保在右侧和颈静脉左侧有足够的空间, 以方便插入30和 #189; G 针用于 FITC-白蛋白溶液的注入. </li>
	<li> 通过使用30和 #189, 静脉注射 (右颈静脉) (3 分钟以上) 与100和 #181; L 10 毫升/千克 FITC-白蛋白; G 针. </li>
	<li> 15 分钟后, 安乐将鼠标斩首, 迅速从头骨上移除先前描述的大脑 13 , 并将其浸入15分钟, 至少10x 的大脑本身的 pre-chilled 戊. </li>
	<li> 将戊冷冻的大脑移动到 pre-chilled 管中, 并将其存储到-80 和 #176; C 冷冻直到组织学切片. </li>
</ol> 4。FITC-白蛋白注入脑组织切片的研究 <ol> <li> 在最佳切削温度 (OCT) 复合物中嵌入冷冻大脑 (见 材料表 ) 在低温切片之前. </li>
	<li> 按顺序将大脑切成20和 #181; m 厚部分, 低温 (请参阅 材料表 ), 并将其安装到显微镜玻璃幻灯片上. </li>
	<li> 使用特定的 anti-laminin 抗体 (见材料表) 执行 FITC-白蛋白/层粘连蛋白 co-staining, 如前所述 6 。
	<ol> <li> 简要地, 在夜间与主抗体 anti-laminin (pAb 1:1000) 孵育切片, 并进行适当的二级抗体共轭 Cy3. </li>
		<li> 片幻灯片, 安装介质包含4和 #39;, 6-diamidino-2-吲 (DAPI) (吸收峰值 = 360 nm; 发射峰值 = 460 nm (请参阅 材料表 ) </li>
		<li> 使幻灯片在4和 #176 中通宵晾干; C 在黑暗中. </li>
		<li> 在荧光显微镜下观察荧光染色 (参见 材料表 ), 在20X 倍放大器上装有两个 FITC (吸收峰值 = 495 nm; 发射峰值 = 525 nm) 和 Cy3 滤镜 (吸收峰值 = 550 nm; 发射峰值 = 570 nm)。
		<ol> <li> 在染色后立即查看样品, 用指甲油固定在角上的片, 并在显微镜下检查, 如上图 (4.3.3). </li>
		</ol> </li> </ol> </li> </ol> 5。设置荧光显微镜和彩色图像采集 <ol> <li> 设置荧光显微镜如下. 
	<ol> <li> 在使用前10分钟打开萤光灯。打开显微镜、连接的计算机和数码相机。运行图像分析软件 (请参见 材料表 ). </li>
		<li> 使用适当的目标来优化信号收集和空间分辨率, 并选择适当的筛选器. </li>
	</ol> </li> <li> 获取彩色图像 <ol> <li> 在图像分析软件中选择 [实时] 命令 (参见 材料表 ), 并为每个滤镜设置最佳光照参数. </li>
		<li> 选择 [冻结] 命令以获得单通道彩色图像并添加缩放栏 (#39; 编辑和 #62; 刻录缩放和 #39;) </li>
		<li> 保存 #39 中的每个图像; tiff 和 #39; 格式. </li>
		<li> 通过选择和 #39; 文件和 #62; 合并通道和 #39, 将通道合并到单个图像中; 并将其保存在 </li>
		<li> tiff 和 #39; 格式 </li>
		<li> 每个脑切片 (每张幻灯片6节) 获取至少四二十四位彩色图片 (2200 和 #181; m x 3400 和 #181; m). </li>
	</ol> </li> </ol> 6。FITC-白蛋白外渗的评价 <ol> <li> 通过使用自由可用的 ImageJ 来分析每个通道的荧光强度 14 , 15 . </li>
	<li> 通过每个图像的总 CY3-Laminin 荧光强度使 FITC 白蛋白的总荧光信号强度正常化, 并将血管外的淤血白蛋白报告为绿色荧光强度. </li>
</ol>

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作者没有利益冲突披露

我们在这里描述的技术主要是用来检测脑疾病状态下的 BBB 渗漏。BBB 功能障碍是越来越多的关注, 作为一个常见的特点, 不同的神经系统疾病4。我们以前使用这种方法来描述在一个罕见的神经退行性疾病的小鼠模型, 如 HD6早期紊乱的 BBB 完整性。
该方法利用了程序的相对简单性和有效性, 提供了准确、可靠的结果。它代表了一个高度敏感的?...

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author&#39;s names was corrected from:
Maglione Vittorio
to:
Vittorio Maglione

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author's names was corrected.

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

中枢神经系统内稳态是神经元细胞正常沟通和功能的先决条件。中枢神经系统的实质是由内皮血脑屏障 (BBB) 与周围紧密封闭的, 它代表着外周血和脑的接口, 在这两个区之间的相声中起着举足轻重的作用1 ,2。血脑屏障是一种复杂的动态三维结构, 主要由专门的微血管内皮细胞 (ECs) 通过细胞间连接-紧密接合 (TJs) 和周, 神经元结尾和星形胶质细胞脚处理1,2。
在生理条件下, 完整的血脑屏障的极低通透性确保了对营养物质和其他分子进出大脑的严格调节, 并为中枢神经系统提供了独特的保护, 使其不会发生可能影响神经活动和反对可能的周边侮辱的血液的构成1,2,3。
长期以来, 对 BBB 完整性及其增强的渗透性的破坏已经成为许多神经和神经退行性疾病的关键特征4包括亨廷顿氏病 (HD)5,6, 但是, 这种功能失调是一种致病现象还是在病程中繁殖事件尚不清楚。血脑屏障破裂的时间也仍然难以捉摸, 然而, 我们组和其他人的新证据表明, 扰乱 bbb 完整性并不代表疾病进展的晚期事件, 而是早期的步骤6,7,8, 可能会产生长期后果。
考虑到这一点, 重要的是要早熟揭示 BBB 故障在神经, 以制定战略有用的预测疾病进展和脑损伤, 并制定替代和更有针对性的干预, 能够成功减轻这种破坏的临床后果。因此, 对脑屏障损害的可靠影像学研究, 在大脑疾病的实验和临床管理中具有重要意义。
本文介绍了利用高分子量荧光素异硫氰酸酯标记白蛋白 (FITC-白蛋白) 在 HD 小鼠模型中评价 BBB 通透性的一种成功而简单的方法。FITC-白蛋白的外渗, 通常不能越过屏障, 进入脑实质被测量为 BBB 渗漏的一个指标。该技术易于适应大鼠和其他病理条件的特点, cerebrovasculature 损害9,10。

<table><tbody><tr><td>Albumin-fluorescin isothiocyanate conjiugate</td><td>SIGMA</td><td>A9771-100MG</td><td></td></tr><tr><td>pAb anti-Laminin</td><td>Novus Biologicals</td><td>NB300-144</td><td></td></tr><tr><td>CY3 anti-rabbit made in goat</td><td>MILLIPORE</td><td>AP132</td><td></td></tr><tr><td>SUPERFROST PLUS</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>Cover Slips 24 X 50 mm</td><td>Thermo Scientific (DIAPATH)</td><td>61050</td><td></td></tr><tr><td>Kilik Optimal Cutting Temperature (OCT) compound</td><td>Bio Optica</td><td>05-9801</td><td></td></tr><tr><td>VECTASHIELD Mounting Media</td><td>VECTOR</td><td>H-1500</td><td>Mounting media with DAPI</td></tr><tr><td>iNSu/Light Insulin Disposible Syringe</td><td>RAYS Health &amp;amp; Safety</td><td>INS1ML26G13</td><td></td></tr><tr><td>30G 1/2&quot;</td><td>BD Microlance</td><td>304000</td><td>Needle for Insulin disposible Syringe</td></tr><tr><td>Scalpel Handle</td><td>F.S.T.</td><td>91003-12</td><td></td></tr><tr><td>#22 Disposable Scalpel blads</td><td>F.S.T.</td><td>10022-00</td><td></td></tr><tr><td>Fine Iris scissors 10.5 cm</td><td>F.S.T.</td><td>14094-11</td><td></td></tr><tr><td>Dumont Forceps #5745 45&amp;deg; 0.10 x 0.06 mm</td><td>F.S.T.</td><td>11251-35</td><td></td></tr><tr><td>Graefe Forceps 10 cm</td><td>F.S.T.</td><td>11051-10</td><td></td></tr><tr><td>Dumont Forceps #5 0.1 X 0.06 mm</td><td>F.S.T.</td><td>11251-20</td><td></td></tr><tr><td>Medical patch</td><td>Medicalis</td><td>34788</td><td></td></tr><tr><td>Sterile disposable towel drape</td><td>Dispotech</td><td>TVO50VE</td><td></td></tr><tr><td>Stereoscopic Microscope</td><td>NIKON</td><td>SMZ 745 T</td><td></td></tr><tr><td>Optic Illuminator LED light (C-FLED2)</td><td>NIKON</td><td>1003167</td><td>Optic Illuminator for Stereoscopic Micrscope</td></tr><tr><td>Eclipse Ni-U Microscope</td><td>Nikon</td><td>932162</td><td>Epifluorescence Microscope</td></tr><tr><td>Microscope digital Camera</td><td>Nikon</td><td>DS-Ri2</td><td>Microscope camera</td></tr><tr><td>Intenslight</td><td>Nikon</td><td>C-HGFI</td><td>Microscope lamp</td></tr><tr><td>NIS-Elements 64 bit</td><td>Nikon</td><td>AR 4.40.00</td><td>Analysis Software</td></tr><tr><td>Electric Razor</td><td>Gemei</td><td>GM-3007</td><td></td></tr></tbody></table>

assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease

破坏血脑屏障 (BBB) 的完整性是一个常见的特点, 不同的神经和神经退行性疾病。虽然扰动 bbb 稳态和脑疾病的发病机制之间的相互作用需要进一步的研究, 开发和验证可靠的程序, 以准确地检测 bbb 改变可能是至关重要的, 并代表了一个有用的工具潜在的预测疾病进展和制定有针对性的治疗策略。
在这里, 我们提出了一个简单和有效的程序, 评估脑屏障渗漏的神经退行性的情况, 如发生在一个前临床小鼠亨廷顿疾病模型, 其中的缺陷, 血 bbb 的渗透性是明确可探测早熟这种疾病。具体来说, 高分子量的荧光素异硫氰酸酯标记 (FITC)-白蛋白, 只有当后者被削弱时才能够穿过 BBB, 在血管或实质区内被剧烈注入小鼠颈静脉并分布然后由荧光显微镜测定。
绿色荧光-白蛋白在脑实质中的积累是异常 BBB 通透性的一个指标, 当定量使用图像 J 处理软件时, 报告为绿色荧光强度。

适当的 FITC-白蛋白注入颈静脉的结果在绿色荧光示踪剂从血液进入大脑 parenchymawhen BBB 是危及6。在生理条件下, 注入的荧光白蛋白的分布仅限于脑血管内, 周围血管区和/或脑实质的信号都是可探测的 (图 1A, 显微顶)。相反, 当血脑屏障在神经病理环境下被破坏时, FITC-白蛋白在血管空间和大脑实质 (图 1A, 底部显?...

Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

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

在这项研究中, 我们提出了一个简单和有效的程序来评估在神经退行性条件下血脑屏障的破坏。为了达到我们的目标, 我们在小鼠颈静脉内注入了高分子荧光素异硫氰酸酯标记 (FITC)-白蛋白, 并通过荧光显微镜将其渗漏到脑实质中。

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 ...

assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc

Research

JoVE Journal

Neuroscience

9.9K 次观看.  IRCCS Neuromed. 在这项研究中, 我们提出了一个简单和有效的程序来评估在神经退行性条件下血脑屏障的破坏。为了达到我们的目标, 我们在小鼠颈静脉内注入了高分子荧光素异硫氰酸酯标记 (FITC)-白蛋白, 并通过荧光显微镜将其渗漏到脑实质中。

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

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透:一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

科研

生物工程

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

医学

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 ...

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

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 ...

通过免疫荧光染色分析血脑屏障模型

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

作者聚焦：推进组织芯片技术在人体组织中的应用 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

<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 ...

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

摘要

探索更多视频

此视频中的章节

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透:一个模型来研究Nanoformulated分子的交付

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

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

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

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

摘要

探索更多视频

此视频中的章节

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透:一个模型来研究Nanoformulated分子的交付

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

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

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模