一种简便、可再生的新分离大鼠脑微血管膜样品制备方法
Summary
本文介绍了一种分离大鼠脑微血管及制备膜样品的方法。该协议具有明显的优势, 生产丰富的微血管样品与可接受的蛋白质产量从个别动物。样品可用于大脑微血管内皮的健壮蛋白质分析。
Abstract
血脑屏障 (BBB) 是一种动态屏障组织, 对各种病理生理和药理刺激反应。这些刺激导致的这种变化可以极大地调节药物向大脑的传递, 并通过扩展, 在治疗中枢神经系统疾病方面造成相当大的挑战。许多影响药物治疗的脑屏障变化涉及蛋白质的局部化和表达在内皮细胞的水平。事实上, 这种关于健康和疾病的脑屏障生理学的知识引起了对这些膜蛋白研究的极大兴趣。从基础科学研究的角度来看, 这意味着需要一个简单但健壮和可重复的方法来分离从实验动物身上采集的脑组织中的微血管。为了从新鲜分离的微血管中制备膜样品, 必须在内皮细胞中丰富样品制剂, 但在神经血管单元的其他细胞类型 (如星形胶质细胞、小胶质、神经细胞、毛细血管)。一个额外的好处是能够从单个动物身上制备样本, 以便在实验人群中捕捉蛋白质表达的真实变异性。本文详细介绍了一种用于大鼠脑微血管分离和制备膜样品的方法。通过使用四离心步骤, 在样品缓冲中加入葡聚糖, 可获得从样品中提取的微血管。该协议可以很容易地由其他实验室为自己的具体应用而调整。从该协议生成的样本已经证明, 从蛋白质分析实验中获得健壮的实验数据, 可以极大地帮助理解对生理、病理生理学和药理刺激的脑屏障反应。
Introduction
血脑屏障 (BBB) 存在于中枢神经系统 (CNS) 与系统循环之间的界面, 在维持脑稳态中起着至关重要的作用。具体地说, 脑屏障功能精确控制大脑胞外液中的溶质浓度, 并有效地提供脑组织所需的养分, 以满足中枢神经系统1的相当大的新陈代谢需求。这些作用意味着脑屏障, 主要存在于微血管内皮细胞的水平, 必须拥有离散机制, 使某些物质能够进入大脑实质, 同时确保潜在的有害外来化合物不能积累。事实上, 脑微血管内皮细胞不是 fenestrated 和表现有限的吞饮, 这确保缺乏非选择性通透性 2.此外, 脑微血管内皮细胞表达紧密连接和 adherens 结蛋白, 在相邻的内皮细胞之间形成物理 “海豹”, 并极大地限制血液传播物质在脑中的旁细胞扩散。实质。事实上, 内源和外源物质的选择性渗透性需要吸收和流出转运体的功能表达式3。总的来说, 紧密的连接, adherens 连接, 和运输工作, 以保持独特的屏障性能的血屏障。
脑屏障是一个动态屏障, 反应生理, 病理生理学和药理刺激。例如, 缺氧/复氧应力已被证明可以调节临界紧致结蛋白 (即occludin、zonulae occluden-1 (ZO-1)) 的表达, 这与增加旁细胞通透性的血管标记有关, 如作为蔗糖4,5,6。在创伤性脑损伤的设置方面也有类似的观察:7和外围炎症疼痛8,9。这些同样的疾病也可以调节在 BBB10,11,12,13,14的传输机制。事实上, 缺氧/复氧损伤增强了有机阴离子转运多肽 1a4 (Oatp1a4) 在脑屏障中的功能表达, 这可能导致特定 Oatp 运输基质的血液到大脑传输显著增加, 如胆酸和阿托伐他汀13。脑屏障的特性也可以通过药物治疗本身来改变, 这种机制可以为大脑和药物相互作用的药物有效性的深刻变化奠定基础。例如, 扑热息痛靶向脑微血管内皮细胞核受体信号机制, 增加临界外排转运蛋白 p-糖蛋白 (p gp) 的功能表达, 并修改时间依赖性镇痛由吗啡、阿片类镇痛药和建立的 P gp 运输基质15授予。彻底了解血脑屏障的变化, 这可能是由疾病或药物引起的, 也需要识别和鉴定特定的调控机制, 控制这些修改。事实上, 在控制紧结蛋白的分子表达的脑微血管内皮细胞中发现了离散信号通路16,17和运输商15, 18,19。这些观察结果表明, 复杂的分子通路参与了对脑屏障紧密连接和转运体的健康和疾病的调控。
在研究脑屏障的一个重大挑战是一个简单有效的方法, 从实验动物的大脑组织中分离微血管和后续制备膜样品的绝对要求。这些样品必须准备好, 使它们既丰富的脑微血管内皮细胞和有限的存在其他细胞类型。在过去的几年中, 科学家在科学文献13、20、21、22中报告了多种从啮齿动物脑中分离微血管的方法。本文介绍了一种简单、健壮、可重现的方法, 用于分离大鼠脑中的微血管, 并制备出一种能用于蛋白质表达分析的内皮膜富集样品。这种微血管隔离协议的优点是能够获得高质量的样品制备, 并能从单个实验动物那里得到足够的蛋白质产量。这样就可以考虑蛋白质表达中动物间的变异。此项协议的进展大大提高了脑屏障研究的健壮性, 因为现在可以避免对脑屏障蛋白变化的真实程度进行过度估计 (或估计不足)。另外, 加入葡聚糖的多重离心步骤, 可以改善实验样品中微血管的富集, 同时有助于去除不必要的细胞成分, 如神经元。
Protocol
Representative Results
Discussion
本文介绍了一种简单有效的从大鼠脑组织中分离的微血管制备膜蛋白样品的方法。在文献13、20、21、22中, 已报告了几种分离大鼠脑微血管和/或生成来自孤立微孔的膜制剂的方法。,24. 虽然上文所述的微血管隔离协议在原理上是相似的, 但这一方法已经得到了优化, 能够制备出…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院 (R01-NS084941) 和亚利桑那生物医学研究委员会 (ADHS16-162406) 提供给 PTR 的赠款的支持。T32-HL007249 已经获得了从博士后任命到国家卫生培训补助金研究所的支持。
Materials
| Protease Inhibitor Cocktail | Sigma-Aldrich | #P8340 | Component of brain microvessel buffer |
| D-mannitol | Sigma-Aldrich | #M4125 | Component of brain microvessel buffer |
| EGTA | Sigma-Aldrich | #E3889 | Component of brain microvessel buffer |
| Trizma Base | Sigma-Aldrich | #T1503 | Component of brain microvessel buffer |
| Dextran (MW 75,000) | Spectrum Chemical Mftg Corp | #DE125 | Dextran used in centrifugation steps to separate microvessels from brain parenchyma |
| Zetamine | MWI Animal Health | #501072 | General anesthetic |
| Xylazine | Western Medical Supply | #5530 | General anesthetic |
| 0.9% saline solution | Western Medical Supply | N/A | General anesthetic diluent |
| Filter Paper (12.5 cm diameter) | VWR | #28320-100 | Used for removal of meninges from brain tissue |
| Centrifuge Tubes | Sarstedt | #60.540.386 | Disposable tubes used for dextran centrifugation steps |
| Pierce™ Coomassie Plus (Bradford) Assay | ThermoFisher Scientific | #23236 | Measurement of protein concentration in membrane preparations |
| Wheaton Overhead Power Homogenizer | DWK Life Sciences | #903475 | Required for homogenization of samples |
| 10.0ml glass mortar and pestle tissue grinder | DWK Life Sciences | #358039 | Required for homogenization of samples |
| Hydrochloric Acid | Sigma-Aldrich | #H1758 | Required for pH adjustment of buffers |
| Bovine Serum Albumin | ThermoFisher Scientific | #23210 | Protein standard for Bradford Assay |
| Standard Forceps | Fine Science Tools | #91100-12 | Used for dissection of brain tissue |
| Friedman-Pearson Rongeurs | Fine Science Tools | #16020-14 | Used for opening skull to isolate brain |
| 50 ml conical centrifuge tubes | ThermoFisher Scientific | #352070 | Used for collection of brain tissue following isolation |
| Glass Pasteur Pipets | ThermoFisher Scientific | #13-678-20C | Used for aspiration of cellular debris following dextran spins |
| Ethanol, anhydrous | Sigma-Aldrich | #459836 | Used for cleaning tissue grinder; diluted to 70% with distilled water |
| Ultracentrifuge tubes | Beckman-Coulter | #41121703 | Used for ultracentrifugation of samples |
References
- Rolfe, D. F., Brown, G. C. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol Rev. 77 (3), 731-758 (1997).
- Brzica, H., Abdullahi, W., Ibbotson, K., Ronaldson, P. T. Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke. J Cent Nerv Syst Dis. 9, 1179573517693802 (2017).
- Ronaldson, P. T., Davis, T. P. Targeting transporters: promoting blood-brain barrier repair in response to oxidative stress injury. Brain Res. 1623, 39-52 (2015).
- Witt, K. A., Mark, K. S., Hom, S., Davis, T. P. Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol. 285 (6), H2820-H2831 (2003).
- McCaffrey, G., et al. Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress. J Neurochem. 110 (1), 58-71 (2009).
- Lochhead, J. J., et al. Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation. J Cereb Blood Flow Metab. 30 (9), 1625-1636 (2010).
- Lucke-Wold, B. P., et al. Bryostatin-1 Restores Blood Brain Barrier Integrity following Blast-Induced Traumatic Brain Injury. Mol Neurobiol. 52 (3), 1119-1134 (2015).
- Campos, C. R., Ocheltree, S. M., Hom, S., Egleton, R. D., Davis, T. P. Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier. Brain Res. , 6-13 (2008).
- Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., Davis, T. P. Transforming growth factor-beta signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain. J Cereb Blood Flow Metab. 29 (6), 1084-1098 (2009).
- Seelbach, M. J., Brooks, T. A., Egleton, R. D., Davis, T. P. Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein. J Neurochem. 102 (5), 1677-1690 (2007).
- Ronaldson, P. T., Finch, J. D., Demarco, K. M., Quigley, C. E., Davis, T. P. Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier. J Pharmacol Exp Ther. 336 (3), 827-839 (2011).
- Pop, V., et al. Early brain injury alters the blood-brain barrier phenotype in parallel with beta-amyloid and cognitive changes in adulthood. J Cereb Blood Flow Metab. 33 (2), 205-214 (2013).
- Thompson, B. J., et al. Hypoxia/reoxygenation stress signals an increase in organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: relevance to CNS drug delivery. J Cereb Blood Flow Metab. 34 (4), 699-707 (2014).
- Tome, M. E., et al. P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain. J Cereb Blood Flow Metab. 36 (11), 1913-1928 (2016).
- Slosky, L. M., et al. Acetaminophen modulates P-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism. Mol Pharmacol. 84 (5), 774-786 (2013).
- Artus, C., et al. The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells. J Cereb Blood Flow Metab. 34 (3), 433-440 (2014).
- Yu, H., et al. Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Calpha signaling pathway in human cerebral microvascular endothelial cells. Exp Ther Med. 14 (5), 4789-4796 (2017).
- Abdullahi, W., Brzica, H., Ibbotson, K., Davis, T. P., Ronaldson, P. T. Bone morphogenetic protein-9 increases the functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier via the activin receptor-like kinase-1 receptor. J Cereb Blood Flow Metab. 37 (7), 2340-2345 (2017).
- Mesev, E. V., Miller, D. S., Cannon, R. E. Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling. Mol Pharmacol. 91 (4), 373-382 (2017).
- Betz, A. L., Csejtey, J., Goldstein, G. W. Hexose transport and phosphorylation by capillaries isolated from rat brain. Am J Physiol. 236 (1), C96-C102 (1979).
- Yousif, S., Marie-Claire, C., Roux, F., Scherrmann, J. M., Decleves, X. Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy. Brain Res. 1134 (1), 1-11 (2007).
- McCaffrey, G., et al. Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo. J Neurochem. 103 (6), 2540-2555 (2007).
- Brzica, H., et al. The liver and kidney expression of sulfate anion transporter sat-1 in rats exhibits male-dominant gender differences. Pflugers Arch. 457 (6), 1381-1392 (2009).
- Ronaldson, P. T., Bendayan, R. HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells. J Neurochem. 106 (3), 1298-1313 (2008).
- Pustylnikov, S., Sagar, D., Jain, P., Khan, Z. K. Targeting the C-type lectins-mediated host-pathogen interactions with dextran. J Pharm Pharm Sci. 17 (3), 371-392 (2014).
- Obermeier, B., Daneman, R., Ransohoff, R. M. Development, maintenance and disruption of the blood-brain barrier. Nat Med. 19 (12), 1584-1596 (2013).
- Abdullahi, W., Davis, T. P., Ronaldson, P. T. Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?. AAPS J. 19 (4), 931-939 (2017).
