用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Tetramethyl Rhodamine (TMR) dextran 3kD | Thermosfisher | D3308 | |
| Fluorescein isothiocyanate (FITC) dextran 3kD | Thermosfisher | D3306 | |
| Ketamine (Ketavet) | Zoetis | ||
| Xylazine (Rompun) | Bayer | ||
| 0.9% Saline | Fresenius Kabi Deutschland GmbH | ||
| 1X PBS | Gibco | 10010-015 | |
| Tissue-tek O.C.T compound | Sakura Finetek | 4583 | |
| 37% Formaldhehyde solution | Sigma | 252549-1L | prepare a 4% solution |
| Bovine Serum Albumin, fraction V | Roth | 8076.3 | |
| Triton X-100 | Sigma | T8787 | |
| rat anti CD31 antibody, clone MEC 13.3 | BD Pharmingen | 553370 | |
| goat anti rat alexa 568 | Molecular Probes | A-11077 | |
| goat anti rat alexa 488 | Molecular Probes | A-11006 | |
| DAPI | Molecular Probes | D1306 | |
| Aqua polymount | Polyscience Inc | 18606 | |
| 21-gauge butterfly needle | BD | 387455 | |
| serum collection tube | Sarstedt | 41.1500.005 | |
| 2mL eppendorf tubes | Sarstedt | 72.695.500 | |
| Kimtech precision wipes tissue wipers | Kimberley-Clark Professional | 05511 | |
| 384-well black plate | Greiner | 781086 | |
| slides superfrost plus | Thermoscientific | J1800AMNZ | |
| PTFE pestle | Wheaton | 358029 | |
| electric overhead stirrer | VWR | VWR VOS 14 | |
| plate reader | Tecan | Infinite M200 | |
| Cryostat | Microm GmbH | HM 550 | |
| Nikon C1 Spectral Imaging confocal Laser Scanning Microscope System | Nikon | ||
| peristaltic perfusion system | BVK Ismatec | ||
| microcentrifuge | eppendorf | 5415R |
References
- Abbott, N. J., Rönnbäck, L., Hansson, E. Astrocyte-endothelial interactions at the blood-brain barrier. Nature reviews. Neuroscience. 7 (1), 41-53 (2006).
- Zhao, Z., Nelson, A. R., Betsholtz, C., Zlokovic, B. V. Establishment and Dysfunction of the Blood-Brain Barrier. Cell. 163 (5), 1064-1078 (2015).
- Obermeier, B., Daneman, R., Ransohoff, R. M. Development, maintenance and disruption of the blood-brain barrier. Nature Medicine. 19 (12), 1584-1596 (2013).
- Devraj, K., Klinger, M. E., Myers, R. L., Mokashi, A., Hawkins, R. A., Simpson, I. A. GLUT-1 glucose transporters in the blood-brain barrier: differential phosphorylation. Journal of neuroscience research. 89 (12), 1913-1925 (2011).
- Banks, W. A. From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery. Nature reviews. Drug discovery. 15 (4), 275-292 (2016).
- Zlokovic, B. V. Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders. Nature reviews. Neuroscience. 12 (12), 723-738 (2011).
- Paganetti, P., Antoniello, K., et al. Increased efflux of amyloid-β peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis. Journal of Alzheimer’s disease: JAD. 38 (4), 767-786 (2014).
- Devraj, K., Poznanovic, S., et al. BACE-1 is expressed in the blood-brain barrier endothelium and is upregulated in a murine model of Alzheimer’s disease. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 36 (7), 1281-1294 (2016).
- Daneman, R. The blood-brain barrier in health and disease. Annals of neurology. 72 (5), 648-672 (2012).
- Gurnik, S., Devraj, K., et al. Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling. Acta neuropathologica. 131 (5), 753-773 (2016).
- Scholz, A., Harter, P. N., et al. Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma. EMBO Molecular Medicine. 8 (1), 39-57 (2016).
- Gross, S., Devraj, K., Feng, Y., Macas, J., Liebner, S., Wieland, T. Nucleoside diphosphate kinase B regulates angiogenic responses in the endothelium via caveolae formation and c-Src-mediated caveolin-1 phosphorylation. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 37 (7), 2471-2484 (2017).
- Ziegler, N., Awwad, K., et al. β-Catenin Is Required for Endothelial Cyp1b1 Regulation Influencing Metabolic Barrier Function. The Journal of neuroscience: the official journal of the Society for Neuroscience. 36 (34), 8921-8935 (2016).
- Vutukuri, R., Brunkhorst, R., et al. Alteration of sphingolipid metabolism as a putative mechanism underlying LPS-induced BBB disruption. Journal of Neurochemistry. , (2017).
- Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis Exp. (65), e3564 (2012).
- Hoffmann, A., Bredno, J., Wendland, M., Derugin, N., Ohara, P., Wintermark, M. High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations. Translational stroke research. 2 (1), 106-111 (2011).
- Armulik, A., Genové, G., et al. Pericytes regulate the blood-brain barrier. Nature. 468 (7323), 557-561 (2010).
- Daneman, R., Zhou, L., Kebede, A. A., Barres, B. A. Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature. 468 (7323), 562-566 (2010).
- Bell, R. D., Winkler, E. A., et al. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron. 68 (3), 409-427 (2010).
- Banks, W. A., Gray, A. M., et al. Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit. Journal of Neuroinflammation. 12, 223 (2015).
- Krause, G., Winkler, L., Mueller, S. L., Haseloff, R. F., Piontek, J., Blasig, I. E. Structure and function of claudins. Biochimica et biophysica acta. 1778 (3), 631-645 (2008).
- Johansson, B. B. Blood-Brain Barrier: Role of Brain Endothelial Surface Charge and Glycocalyx. Ischemic Blood Flow in the Brain. , 33-38 (2001).
- Fu, B. M., Li, G., Yuan, W. Charge effects of the blood-brain barrier on the transport of charged molecules. The FASEB Journal. 22 (1 Supplement), (2008).
- Goebl, N. A., Babbey, C. M., Datta-Mannan, A., Witcher, D. R., Wroblewski, V. J., Dunn, K. W. Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells. Molecular biology of the cell. 19 (12), 5490-5505 (2008).
- Lopez-Quintero, S. V., Ji, X. -. Y., Antonetti, D. A., Tarbell, J. M. A three-pore model describes transport properties of bovine retinal endothelial cells in normal and elevated glucose. Investigative ophthalmology & visual science. 52 (2), 1171-1180 (2011).
- Hallmann, R., Mayer, D. N., Berg, E. L., Broermann, R., Butcher, E. C. Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier. Developmental dynamics: an official publication of the American Association of Anatomists. 202 (4), 325-332 (1995).
