在缺血样条件下监测星形胶质细胞的反应性和增殖体外
Summary
缺血性中风是一个复杂的事件, 其中星形胶质细胞对受氧葡萄糖剥夺 (OGD) 影响的脑区的具体贡献是很难研究的。本文介绍了一种获取孤立星形胶质细胞的方法, 并研究了其在 OGD 条件下的反应性和增殖。
Abstract
缺血性中风是一种复杂的脑损伤, 血栓或栓子阻碍血液流向大脑的部分。这导致了缺氧和葡萄糖的匮乏, 导致能量衰竭和神经元死亡。在缺血性中风的侮辱, 星形胶质细胞成为反应和增殖周围的伤害现场, 因为它的发展。在这种情况下, 很难研究星形胶质细胞对缺血脑区的特定贡献。因此, 本文介绍了一种方法, 研究原星形胶质细胞的反应性和增殖在一个体外模型的缺血样环境, 称为氧葡萄糖剥夺 (OGD)。星形胶质细胞是从1-4 岁的新生大鼠中分离出来的, 并通过胶质纤维酸性蛋白 (胶质细胞) 和细胞核染色对非特异性星的数量进行评估。星形胶质细胞受 OGD 条件的时间可以定制, 以及他们所接触到的氧气的百分比。这种灵活性允许科学家在不同的细胞组体外描述缺血样状态的持续时间。本文讨论了诱导星形胶质细胞反应性、肥大形态学和增殖细胞核抗原 (PCNA) 免疫荧光法测定 OGD 的时间范围。星形胶质细胞除增殖外, 还具有能量和氧化应激, 并通过释放可溶性因子而对 OGD 反应。这种培养基可以收集并用于分析星形胶质细胞释放的分子在没有细胞相互作用的原代神经细胞中的作用。总之, 这种原细胞培养模型可以有效地用于了解孤立星形胶质细胞在损伤中的作用。
Introduction
中风被定义为 “急性神经功能障碍的血管来源, 无论是突然或快速发展的症状和体征, 对应于大脑中的重点领域的参与”1,2。有两种类型的中风: 出血和缺血性。当血管功能障碍是由动脉瘤或动静脉故障引起的, 伴有动脉后破裂的减弱, 这称为出血性中风3 , 在大多数情况下, 导致死亡。当血栓或栓子阻碍血流, 导致暂时剥夺氧气和葡萄糖的大脑区域, 它被称为缺血性中风4。不滋养细胞周围的受影响地区或缺血核心导致了自我平衡和代谢失衡, 精力充沛的功能障碍, 神经元死亡和炎症5, 这可能导致 life-long 残疾患者的6。
缺血性中风是一种多因素损伤, 涉及多种类型的细胞, 在不同的时间点反应和发挥作用。许多相互作用为研究单个细胞的行为创造了一个困难的环境。那么, 我们如何研究一个特定的细胞类型在如此复杂的环境下的贡献呢？一个接受的体外模型的缺血包括暴露细胞到氧和葡萄糖剥夺 (OGD), 在一定时期内, 然后恢复细胞到常环境。该系统模拟缺血性中风后再灌注血。在这种方法中, 细胞或组织被暴露在一个无糖的介质中, 在一个净化氧气的环境中, 使用一个专门的缺氧室。OGD 潜伏期可以从几分钟到24小时不等, 这取决于要测试的假说。研究表明, 根据 OGD 和常环境的时间, 可以实现中风的特定表型 (即、急性或亚慢性)。主要孤立星形胶质细胞, 暴露于 OGD 后恢复到常条件, 是一个研究细胞模型模拟中风体外7。利用 OGD 可以在脑卒中的环境下揭示孤立细胞的独立分子机制。
随着我们对星形胶质细胞生物学知识的增加, 它已经变得明显, 它们对于维持突触和维持神经修复、发育和可塑性是至关重要的8。在正常情况下, 星形胶质细胞释放和反应的因子, 因子, 生长因子和 gliotransmitters, 保持代谢平衡和内稳态的突触5,9。在急性 neuroinflammation, 如缺血性中风, 这些细胞可以成为反应, 显示长期过度表达的胶质纤维酸性蛋白 (胶质), 并显示肥大在其形态学5,10,11,12. 随着缺血性梗塞的发展, 星形胶质细胞所提供的稳态对正常的谷氨酸摄取、能量代谢、活性分子的交换和抗氧化活性的影响, 如13。
重新激活的星形胶质细胞在梗塞组织周围增殖, 而白细胞则向损伤区域14迁移。星增殖可以用标记物如增殖细胞核抗原 (PCNA)、Ki67 和尿 (BrdU)15来测量。这种增殖反应是以时间为根据的方式产生的, 它有助于形成胶质瘢痕, 一组不可逆转的反应性星形胶质细胞沿受损部位的软组织损伤后9。这个疤痕的初始功能之一是限制免疫细胞外渗从这个地区。然而, 研究表明, 疤痕成为一个物理障碍的轴突延长, 因为他们释放分子抑制轴突生长, 并创建一个物理屏障, 防止轴突在受伤地区的延伸16。然而, 有科学证据表明, 脊髓损伤后, 完全防止胶质瘢痕形成会损害轴突的再生17。因此, 必须在所研究的伤害的框架内考虑具体的星反应的背景。
所提出的方法可用于研究星形胶质细胞在缺氧性葡萄糖剥夺后的个体化功能, 并可根据研究者想要回答的问题进行修正。例如, 除了形态学变化和在不同 OGD 时间表达的标记, 清星形胶质细胞暴露于 OGD 可以进一步分析, 以确定这些细胞释放出的可溶性因子, 或作为条件介质来评估其在其他脑细胞中的作用。这种方法可以研究星形胶质细胞的反应性, 这可能导致解释的因素, 控制和调节他们的反应在缺血性中风的情况。
Protocol
产后大鼠 (大) 1-4 天后被用来分离皮质。安乐死的方法是斩首, 由 NIH 的指南批准. 1. 准备手术用的仪器和材料 灭菌釜中的仪器 (温度: 121 和 #186; C、压力:15 psi, 时间:30 分钟) 使用钢盒或即时密封灭菌袋。请参阅 材料表 中的材料. 2。完整的 DMEM 准备 在包含700毫升蒸压水的一公升烧杯中, 在室温下添加 Dulbecco 和 #39 ?…
Representative Results
主要的关注之一星文化是存在的其他细胞, 如神经元, 突, 成纤维细胞, 和小胶质。在图 1中, 从大鼠皮层分离出来的细胞每3天就有一次改变, 或者未经治疗或在 1 h. 24 小时后被添加 LME, 细胞被 immunostained 为胶质蛋白和 1-4 DAPI。未经处理的细胞显示平均39% 非胶质细胞阳性, 而 LME 治疗的细胞显示8%。这些结果表明, LME 的治疗和媒介的变化有效地减少了非胶…
Discussion
本协议描述了从大鼠皮层星形胶质细胞的分离。在这种方法, 这是至关重要的, 以减少污染与其他细胞类型, 如小胶质, 突, 和成纤维细胞。为了减少小胶质细胞的数量, 可以采取几个步骤: 改变介质, 轨道震动和化学处理。一旦通过选择性细胞标记或最突出的细胞污染物, 通过免疫荧光证实了培养纯度, 就可以进行实验。例如, 抗电离钙结合剂分子 1 (Iba-1) 抗体可以用来检测小胶质细胞。神经元死亡在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢 Paola 洛佩斯 Pieraldi 的技术援助。A.H.M. 感谢8G12MD007600 和 U54-NS083924 支持这一出版物的赠款。我们感谢 NIH-NIMHD-G12-MD007583 赠款的设施支持。d.e.r. 对 NIHNIGMS-R25GM110513 提供的奖学金表示感谢。我们感谢使用通用仪器区和 Dr. Priscila 加西亚萨纳布里亚的援助, 以利用 RCMI 方案的光学成像设施的赠款 G12MD007583。此外, 我们要感谢何塞帕迪拉在拍摄和编辑视觉协议方面的杰出作用。
Materials
| Instruments for Surgery – Step 1 | |||
| Operating scissor 5.5” | Roboz Company | RS-6812 | Tools used to decapitate the rats. |
| Curved forceps 7” | Roboz Company | RS-5271 | Holds the skin of the rat while the skull is removed. |
| Micro-dissecting scissors 4” | Roboz Company | RS-5882 | Cuts both the skin and skull of the rat. |
| Micro-dissecting forceps 4” angled, fine sharp | Roboz Company | RS-5095 | Holds the skin of the rat while the skull is removed. |
| Micro-dissecting forceps 4” slightly curved 0.8 | Roboz Company | RS-5135 | Tool used to separate cortices. |
| Micro-dissecting tweezers | Roboz Company | RS-4972 | Peels brain meninges. |
| Dissection microscope | Olympus | SZX16 | Important for removing the meninge from the cortices. |
| DMEM Preparation – step 2 | |||
| Dulbecco’s Modified Eagle’s Medium (DMEM) | GibCo. Company | 11995-065 | Supports the growth of cells. |
| Sodium bicarbonate | Sigma-Aldrich Company | S7277 | Supplement for the cell culture media. |
| Fetal bovine serum (FBS) | GibCo. Company | 10437-010 | Serum-supplement for the cell culture. |
| Penicillin-Streptomycin | GibCo. Company | 15140-148 | Inhibits the growth of bacterias in the cell culture. |
| Filter System 1L with 0.22um pore | Corning | 431098 | |
| Astrocyte culture – step 3 | |||
| Serological pipets 5mL | VWR | 89130-896 | To pipette DMEM to containers with cells. |
| Serological pipets 10mL | VWR | 89130-898 | To pipette DMEM to containers with cells. |
| Serological pipets 25mL | VWR | 89130-900 | To pipette DMEM to containers with cells. |
| Centrifuge conical tube 15mL | Santa Cruz Biotechnology | sc-200250 | |
| Safe-lock tube 1.5mL | Eppendorf | 022363204 | |
| Barrier Tips 200 uL | Santa Cruz Biotechnology | sc-201725 | |
| Barrier Tips 1 mL | Santa Cruz Biotechnology | sc-201727 | |
| Biohazard Orange Bag 14 x 19" | VWR | 14220-048 | |
| 60mm petri dishes | Falcon | 351007 | |
| Sterile gauze pads | Honeywell Safety | 89133-086 | |
| Stomacher 80 Biomaster | Sewar Lab System | 030010019 | Triturate the brain tissue. |
| Stomacher 80 Blender Sterile Bags | Sewar Lab System | BA6040 | Sterile bag for the stomacher cell homogenizer. |
| Beaker 400mL | Pyrex | 1000 | |
| Sterile cell dissociation sieve, mesh #60 | Sigma-Aldrich Company | S1020 | To obtain a uniform single cell suspension. |
| Sterile cell dissociation sieve, mesh #100 | Sigma-Aldrich Company | S3895 | To obtain a uniform single cell suspension. |
| Invert phase microscope | Nikon | Eclypse Ti-S | Verify cells for contamination or abnormal cell growth. |
| 75cm2 sterile flasks | Falcon | 353136 | |
| Multi-well plate | Falcon | 353046 | |
| Micro cover glasses (coverslips), 18mm, round | VWR | 48380-046 | |
| Bright-Line hemacytometer | Sigma-Aldrich Company | Z359629 | |
| Pasteur pipettes | Fisher Scientific | 13-678-20D | |
| Ethyl alcohol | Sigma-Aldrich Company | E7023 | |
| L-leucine methyl ester hydrochloride 98% (LME) | Sigma-Aldrich Company | L1002 | Promotes the elimination of microglia cells in the primary cortical astrocyte cultutre. |
| Cytosine β-D-arabinofuranoside (Ara-C) | Sigma-Aldrich Company | C1768 | |
| Poly-D-Lysine Hydrobromide, mol wt 70,000-150,000 | Sigma-Aldrich Company | P0899 | |
| Trypsin/EDTA | GibCo. Company | 15400-054 | |
| Trypan Blue | Sigma-Aldrich Company | T8154 | |
| Phosphate buffer saline (PBS) tablets | Calbiochem | 524650 | |
| Sterile Water | Sigma-Aldrich Company | W3500 | |
| OGD Medium Preparation – step 5 | |||
| Centrifuge conical tube 50 mL | VWR | 89039-658 | |
| Dulbecco’s modified Eagle’s medium-free glucose | Sigma-Aldrich Company | D5030 | Supports the growth of cells. |
| Sodium bicarbonate | Sigma-Aldrich Company | S7277 | Supplement for the cell culture media. |
| Penicillin-Streptomycin | GibCo. Company | 15140-148 | Inhibits the growth of bacterias in the cell culture. |
| 200mM L-glutamine | GibCo. Company | 25030-081 | Amino acid that supplements the growth of cells. |
| Phospahet buffer saline (PBS) tablets | Calbiochem | 524650 | |
| Filter System 50mL with 0.22um pore | Corning | 430320 | |
| Centrifuge conical tube 50 mL | VWR | 89039-658 | |
| Single Flow Meter | Billups-Rothenberg | SMF3001 | Measure gas flow in oxygen purge. |
| Hypoxia Incubator Chamber | StemCell | 27310 | Generates a hypoxic environment for the cell culture. |
| Traceable Dissolved Oxygen Meter | VWR | 21800-022 | |
| 95% N2/ 5% CO2 Gas Mixture | Linde | Purges the environment of oxygen. | |
| primary astrocyte immunofluorescence – step 6 | |||
| Phosphate buffer saline (PBS) tablets | Calbiochem | 524650 | |
| Formaline Solution Neutral Buffer 10% | Sigma-Aldrich | HT501128 | Solution used to fix cells. |
| Methanol | Fisher | A4544 | Solution used to fix cells. |
| Non-ionic surfactant (Triton X-100) | Sigma-Aldrich | T8787 | |
| Fetal bovine serum (FBS) | GibCo. Company | 10437-010 | Serum-supplement for the cell culture. |
| Anti-NeuN | Cell Signaling | 24307 | Detects mature neurons, serves to validate the astrocytic culture. |
| Anti-PCNA | Cell Signaling | 2586 | Detects proliferating cells. |
| Propidium Iodide (PI) | Sigma-Aldrich Company | P4170 | Apoptosis staining. |
| Anti-Olig1 | Abcam | AB68105 | Detects mature oligodendrocytes. |
| Anti-Iba1+ | Wako | 016-20001 | Detects microglial cells. |
| Anti-GFAP Conjugated with Cy3 | Sigma-Aldrich Company | C9205 | Detects reactive astrocytes in the treated cells. |
| Alexa Fluor 488 | Molecular Probe Life Technology | A1101 | Anti-Mouse Secondary Antibody |
| Alexa Fluor 555 | Molecular Probe Life Technology | A21428 | Anti-Rabbit Secondary Antibody |
| 4’,6’-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich Company | D9542 | Nuclear staining |
| Confocal microscope | Olympus |
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Citer Cet Article
Ferrer-Acosta, Y., Gonzalez-Vega, M. N., Rivera-Aponte, D. E., Martinez-Jimenez, S. M., Martins, A. H. Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions. J. Vis. Exp. (128), e55108, doi:10.3791/55108 (2017).