在五个脊椎动物群中可靠隔离中枢神经系统微血管
Summary
该协议的目的是从脑血管和陀螺脊椎动物的中枢神经系统的多个区域分离微血管。
Abstract
从中枢神经系统 （CNS） 分离微血管通常通过结合多只动物（最常见的是啮齿动物）的皮质组织来执行。这种方法将血脑屏障（BBB）特性的询问限制在皮层，不允许进行个体比较。该项目的重点是开发一种隔离方法，允许比较来自多个中枢神经系统区域的神经血管单元（NVU）：皮层、小脑、视叶、下丘脑、脑干和脊髓。此外，最初为鼠类样本开发的这个方案被成功应用于中小脊椎动物的CNS组织，从中我们也能够从大脑半球白质中分离微血管。当与免疫标记配对时，该方法允许对蛋白质表达进行定量，并在个体、组织类型或治疗之间进行统计比较。通过评估实验性自身免疫性脑脊髓炎 （EAE） 期间蛋白质表达的变化，我们证明了这种适用性，这是神经炎疾病多发性硬化症的一种小鼠模型。此外，通过这种方法分离的微容器可用于下游应用，如qPCR、RNA-seq和西体斑点等。尽管这不是第一次尝试在不使用超离心或酶分离的情况下分离CNS微容器，但它在单一个体和多个CNS区域的比较方面的独特之处。因此，它允许调查一系列差异，否则可能仍然模糊： 中枢神经系统部分 （皮质， 小脑， 视叶， 脑干， 下丘脑， 脑垂体， 和脊髓）， CNS 组织类型 （灰色或白色物质）， 个人，实验性治疗组和物种。
Introduction
我们的大脑是我们身体中最重要的器官。因此，保持大脑平衡，尽管外部因素，可能会触发偏离正常是一个优先事项。据一些学者说，大约4亿至5亿年前，脊椎动物发展了我们现在所知的血脑屏障（BBB）2，3。这种保护性”篱笆”通过严格调节血液和中枢神经系统之间的离子、分子和细胞的传输，对中枢神经系统 （CNS） 平衡和功能产生最大的影响。当BBB被打乱时，大脑变得易受毒性暴露、感染和炎症的影响。因此，BBB功能障碍与许多，如果不是全部，神经和神经发育障碍4，5，6相关。
BBB的精密功能归因于神经血管单元（NVU）2，3所符合的独特CNS微血管。高度专业化的内皮细胞、围细胞和星形端脚是NVU2，3的细胞成分。这些细胞产生的细胞外基质对NVU和BBB生理学也至关重要。虽然NVU的基本细胞和分子成分在脊椎动物中保存，但各阶和物种7、8之间报告存在异质性。然而，技术限制阻碍了我们在神经生物学、生物医学或转化研究方面充分考虑这些差异的能力。
因此，我们扩展了CNS区域特定的微容器隔离方法，使其适用于来自所有五个脊椎动物群的众多物种：鱼类、两栖动物、爬行动物、鸟类和哺乳动物。该协议被描述为用于小脑脑和大陀螺脊椎动物，包括具有翻译相关性的物种9。此外，我们包括中枢神经系统的其他地区以前没有在此范围内调查，但与神经生理学相关，具有巨大的临床影响：下丘脑，脑垂体和白质。最后，我们测试了这种分离方法作为识别沿NVU和/或BBB9、10、11的蛋白质表达变化的可靠工具的能力。作为概念验证，我们展示了如何使用免疫荧光之后的隔离方法确定 EAE 期间 VCAM-1 和 JAM-B 表达的变化。
Protocol
本研究的所有程序均符合加州大学（UC）、戴维斯机构动物护理和使用委员会（IACUC）制定的指导方针。加州大学戴维斯分校的动物护理由多个独立资源监管，自1966年以来一直获得国际实验室动物护理评估与认证协会（AAALAC）的认证。猪中枢神经系统组织从UCD动物科学系，肉类科学实验室获得。来自河河猴的CNS组织从加州国家灵长类研究中心病理学部（NIH P51OD0111107）获得。实验室工作人员对猪和?…
Representative Results
从鼠中枢分离的微血管显示神经血管单元2，3的所有内在细胞成分。使用血小板内皮细胞粘附分子-1（PECAM，也称为CD31）或同化素IB4（一种结合内皮细胞的糖蛋白，用于内皮细胞， 血小板衍生的生长因子-β（PDGFR®）或神经元胶质抗原2（NG2）用于围细胞和水孢素-4（AQP4），用于星体端脚（图3A，B），</…
Discussion
BBB包括脑微血管内皮细胞的独特特性，加上复杂的结构，紧，粘附，”粘接”结，和粘附斑块关键CNS平衡2，3，19。内皮细胞的特性是由围细胞和周围的星体末端脚过程2，3，19诱导和维护。BBB微血管显示极性（即，在发光或腹皮细胞表面局部的蛋白质的不对称?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Cruz-Orengo博士得到了加州大学戴维斯分校兽医学院启动基金的支持。
Materials
| 10X PBS | ThermoFisher | BP39920 | Used for blocking and antibody diluent. |
| 20% PFA | Electron Microscopy Sciences | 15713-S | Used as fixative (4% PFA) |
| 70,000 MW Dextran | Millipore Sigma | 9004-54-0 | Used for MV-2 solution |
| Adson Forceps | Fine Science Tools (FST) | 11006-12 | Used for removal of muscle and skin |
| Adson Forceps, student quality | FST | 91106-12 | Same as above but cheaper |
| Bovine serum albumin (BSA) | Millipore Sigma | A7906-100G | Used for MV-3 solution, blocking and antibody diluent |
| Corning 100 μm Cell strainer | Millipore Sigma | CLS431752-50EA | |
| Corning 70 μm Cell strainer | Millipore Sigma | CLS431751-50EA | |
| Corning Deskwork low-binding tips | Millipore Sigma | CLS4151 | Same as below but cheaper. |
| Cultrex Poly-D-Lysine | R&D | 3439-100-01 | Used for slide coating |
| Donkey anti-Goat IgG-ALEXA 555 | Thermo | A21432 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Mouse IgG-ALEXA 488 | Thermo | A21202 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Rabbit IgG-ALEXA 488 | Thermo | A21206 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Rabbit IgG-ALEXA 647 | Thermo | A31573 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Rat IgG-DyLight 650 | Thermo | SA5-10029 | Used as secondary antibody. Recommended dilution of 1:200. |
| Double-Pronged Tissue Pick | FST | 18067-11 | Used for removal of meninges and choroid plexus |
| Dumont #3c Forceps | FST | 11231-20 | Used for more delicate and/or small CNS tissue handling (like pituitary) |
| Dumont #7 Forceps | FST | 11274-20 | Used for CNS tisssue dissection and handling |
| Dumont #7 Forceps, student | FST | 91197-00 | Same as above but cheaper |
| ep Dualfilter T.I.P.S. LoRetention Tips | Eppendorf | 22493008 | Better quality than the tips above (more expensive). |
| Extra Fine Graefe Forceps, serrated | FST | 11151-10 | Used for bone removal |
| Fine Scissors, sharp | FST | 14060-09 | Used for removal of pig and macaque dural sac |
| Glass Pestle 1.5 mL Microcentrifuge Tube Tissue Grinder Homogenizer, Pack of 10 | Chang Bioscience Inc. (eBay) | GP1.5_10 | Used for small vetebrate hypothalus and pituitary. |
| Goat anti-CXCL12, biotinylated | PeproTech | 500-P87BGBT | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution: 1:20. |
| Goat anti-JAM-B | R&D | AF1074 | Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 μg/mL. |
| Goat anti-Mouse IgG-ALEXA 488 | Thermo | A11001 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-Mouse IgG-ALEXA 555 | Thermo | A21424 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-PDGFRβ | R&D | AF1042 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Goat anti-Rabbit IgG-ALEXA 555 | Thermo | A21249 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-Rabbit IgG-DyLight 488 | Thermo | 35552 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-Rat IgG-DyLight 650 | Thermo | SA5-10021 | Used as secondary antibody. Recommended dilution of 1:200. |
| Graefe Forceps, curved tip, 1X2 teeth | FST | 11054-10 | Use for nylon filter net holding and shaking |
| HBSS, 1X buffer with calcium and magnesium | Corning | 21-022-CM | Used for MV-1 solution |
| HEPES, 1M liquid buffer | Corning | 25-060-CI | Used for MV-1 solution |
| Isolectin GS-IB4-Biotin-XX | ThermoFisher Scientific (Thermo) | I21414 | Glycoprotein isolated from legume Griffonia simplicifolia that binds D-galactosyl residues of endothelial cell glycocalysx. Used for avian and porcine CNS microvessels. Recommended concentration: 5 μg/mL. |
| LaGrange Scissors, serrated | FST | 14173-12 | Used for skull dissection and laminectomy (except pig and macaque) |
| Millicell EZ slide 8-well unit | Millipore Sigma | PEZGS0816 | |
| Mouse anti-CLDN5 | Thermo | 35-2500 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Mouse anti-GGT1 | Abcam | ab55138 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Mouse anti-Human CD31 | R&D | BBA7 | Used as primary antibody on primate CNS microvessels. Recommended concentration: 16.5 μg/mL. |
| Mouse anti-NFM | Thermo | RMO-270 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Mouse anti-αSMA | Thermo | MA5-11547 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Nylon Filter Net, roll | Millipore Sigma | NY6000010 | Laser-cut to 13 mm diameter filter net discs. Used for small vetebrate hypothalus and pituitary. |
| Nylon Filter Nets, 25 mm | Millipore Sigma | NY2002500 | Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary. |
| Nylon Filter Nets, 47 mm | Millipore Sigma | NY2004700 | Used for macaque and pig CNS tissues, except hypothalamus and pituitary. |
| ProLong Gold antifade reagent with DAPI | ThermoFisher | P36935 | Used to coverslip slides. |
| Rabbit anti-AQP4 | Millipore Sigma | A5971 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Rabbit anti-LSR | Millipore Sigma | SAB2107967 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Rabbit anti-NG2 | Millipore Sigma | AB5320 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Rabbit anti-OSP | Abcam | ab53041 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 1 μg/mL. |
| Rabbit anti-VE-Cadherin | Abcam | ab33168 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Rabbit anti-ZO-1 | Thermo | 61-7300 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Rat anti-CD31 | Becton Dickinson | BD 550274 | Used as primary antibody for murine CNS microvessels. Recommended concentration: 5 μg/mL. |
| Rat anti-GFAP | Thermo | 13-0300 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Rat anti-VCAM-1 | Becton Dickinson | BD 553329 | Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 μg/mL. |
| Sterile Ringer's Solution, Frog | Aldon Corporation | IS5066 | Used for amfibian anesthesia |
| Streptavidin-ALEXA 555 | Thermo | S32355 | Used as secondary antibody to label biotinylated primary antibodies. Recommended dilution of 1:500. |
| Streptavidin-ALEXA 647 | Thermo | S32357 | Used as secondary antibody to label biotinylated primary antibodies. Recommended dilution of 1:500. |
| Surgical Scissors, sharp | FST | 14002-12 | Used for removal of muscle and skin |
| Surgical Scissors, sharp-blunt | FST | 14001-16 | Used for decapitation (except pig and macaque) |
| Swinnex Filter Holder, 13 mm | Millipore Sigma | SX0001300 | Modified by laser-cut. Used for small vetebrate hypothalus and pituitary. |
| Swinnex Filter Holder, 25 mm | Millipore Sigma | SX0002500 | Modified by laser-cut. Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary. |
| Swinnex Filter Holder, 47 mm | Millipore Sigma | SX0004700 | Modified by laser-cut. Used for macaque and pig CNS tissues, except hypothalamus and pituitary. |
| Triton X-100 | ThermoFisher | 50-165-7277 | Used for blocking and antibody diluent. |
| Wheaton 120 Vac Overhead Stirrer | VWR (Supplier DWK Life Sciences) | 62400-904 (DWK #903475) | Used for macaque and pig CNS tissues with 55 mL tissue grinder, except hypothalamus and pituitary. |
| Wheaton Potter-Elvehjem tissue grinder with PTFE pestle, 10 mL | VWR (Supplier DWK Life Sciences) | 14231-384 (DWK #357979) | Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary. |
| Wheaton Potter-Elvehjem tissue grinder with PTFE pestle, 55 mL | VWR (Supplier DWK Life Sciences) | 14231-372 (DWK #357994) | Used for macaque and pig CNS tissues, except hypothalamus and pituitary. |
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Cite This Article
Yuan, Y., Dayton, J. R., Freese, M., Dorflinger, B. G., Cruz-Orengo, L. Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups. J. Vis. Exp. (155), e60291, doi:10.3791/60291 (2020).