性别差异在小鼠海马星形胶质细胞<em>在体外</em>缺血
Summary
星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。在这里，我们报告，以研究男性和女性新生儿幼崽的星形胶质细胞功能的体外缺血后背后的机制性控海马星形胶质细胞培养协议的实用方法。
Abstract
星形胶质细胞增生缺氧/缺血（HI）相关的脑损伤起着新生儿增加发病率和死亡率的作用。最近的临床研究表明，大脑损伤的严重程度似乎是性别依赖性的，并且该男性新生儿是HI有关的脑损伤的影响更加敏感，导致更严重的神经系统的结果相比，女性具有可比的脑损伤。可靠的方法来隔离和维护绝育手术海马星形胶质细胞的高度浓缩的人口的发展是至关重要的了解新生儿HI的病理后果的性别差异细胞基础。在这项研究中，我们描述了创建经受体外缺血，缺氧缺糖模型，随后复氧性具体海马星形细胞培养的方法。随后的反应性星形胶质被immunostai检查宁为胶质纤维酸性蛋白（GFAP）和S100B。这种方法提供了一个有用的工具来研究男性和女性的海马星形胶质细胞的新生之后HI的作用，分别。
Introduction
星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。越来越多的证据表明，星形胶质细胞的作用是比提供支持神经元多。事实上，在生理条件下的星形胶质细胞的作用可以是非常复杂的，如引导显影轴突1的迁移，调节CNS血流2中，并保持突触组织液3的pH稳态，和参与血脑屏障4和突触传递5。在病理条件下，星形细胞损伤响应与被叫其中形态，数量，位置，地形（相对于从损伤的距离）的星形胶质细胞和功能可在异构方式6,7-改变反应性星形胶质细胞增生的过程。看到下面的星形胶质细胞增生新生儿缺氧缺血性脑病，也许有利于新生儿的发病率和死亡率<s达> 8。
最近的临床和实验研究表明，大脑损伤的严重程度似乎是性别依赖性和，作为相对于男性新生儿对缺氧/缺血（HI）相关的脑损伤的影响更加敏感，导致更严重的神经成果女性具有可比性脑损伤9-11。虽然损伤的定位取决于胎龄和持续时间和损伤的严重程度，海马是足月HI后在CNS中最常见的影响区域中的一个，并增加海马星形胶质细胞增生被上调的确认胶质纤维酸性蛋白（GFAP）新生儿HI 7,10,12,13后3天。星形胶质细胞功能的性别差异被证明在这两个新生儿和成人啮齿动物脑缺血后14,15。另外，男性星形细胞易感性的体外缺血通过增加CEL所示升死亡相比，在文化16女性皮层星形胶质细胞。
性别差异在子宫内开始并持续直至死亡17。在过去十年中，包括在细胞培养和体内研究，在实验条件下男女的重要性一直医学和NIH研究所的重点寻求基础知识，在生理和病理条件17,18见过的性别差异。可靠的方法开发分离和维护绝育手术海马星形胶质细胞的人口有必要了解的新生儿HI的病理后果的性别差异的细胞学基础。本研究的目的是提供的技术准备，以评估以下氧气/葡萄糖剥夺（OGD）和复氧（再氧化），诱导GFAP免疫反应阳性星形胶质细胞的作用，从新生小鼠丰富的性别特异性海马星形胶质细胞培养HI在细胞培养环境。这种技术可以用来测试有关含氧量正常和缺血性的条件下在新生儿男性和女性海马星形胶质细胞的任何假设。
Protocol
Representative Results
Discussion
为了研究生理和病理条件下，在属性的性别差异和功能星形胶质细胞，在细胞培养物中制备性控初级星形胶质细胞是利用一个重要工具。在本研究中，我们报道了高效和可再现的方法来培养新生性控海马星形胶质细胞的高度富集的同质群体（P0-P2）的C57BL / 6（野生型）或K19F（GFAP空）小鼠幼仔体外 。建立这种方法可以帮助研究者了解性控海马星形胶质细胞的生理和病理功能体外缺血?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Clinical and Translational Science Award program of NCATS UL1 TR0000427 and KL2 TR000428 (Cengiz P), UL1TR000427 to the UW ICTR from NIH/NCATS and funds from Waisman Center (Cengiz P), K08 NS088563-01A1 from NINDS (Cengiz P) and NIH P30 HD03352 (Waisman Center), NIH/NINDS 1K08NS078113 (Ferrazzano P). We would like to thank Albee Messing, PhD, for providing us the GFAP knockout mice.
Materials
| Astrocyte culture media | |||
| DMEM, high glucose | cellgro | 10-013-CV | |
| Horse serum | Gibco | 26050-070 | Final Concentration: 10% |
| Penicillin-Streptomycin | Cellgro | 30-002-CI | Final Concentration: 1% |
| L-Leucine methyl ester hydrochloride | Aldrich | L1002-25G | Final Concentration: 5 mM |
| Solution for brain tissue digestion | |||
| HBSS | Life Technologies | 14170-088 | |
| 0.25% Trypsin | cellgro | 25-050-CI | Final Concentration: 0.25% |
| Outro | |||
| 70% (vol/vol) ethanol | Roth | 9065.2 | |
| Poly-D-Lysine 12mm round coverslips | Corning | 354087 | |
| Water | Sigma | W3500 | cell culture grade |
| PBS | cellgro | 21-040-CV | cell culture grade |
| 0.05% Trypsin-EDTA | Life Technologies | 25300-062 | |
| 70 μm Sterile cell strainer | Fisher scientific | 22363548 | |
| 3.5 cm petri dish | BD Falcon | 353001 | |
| 15 ml Falcon tube | BD Falcon | 352096 | |
| 50 ml Falcon tube | BD Falcon | 352070 | |
| Forceps, fine | Dumont | 2-1032; 2-1033 | # 3c; # 5 |
| Forceps, flat tip | KLS Martin | 12-120-11 | |
| 13 cm surgical scissors | Aesculap | BC-140-R | |
| Confocal Microscope | Nikon | A1RSi | |
| Centrifuge | Eppendorf | 5805000.017 | Centrifuge5804R |
| Orbital Shaker | Thermo Scientific | SHKE 4450-1CE | MaxQ 4450 |
| Anti-IBA1 | Wako | 019-19741 | Rabbit monoclonal |
| Anti-MAP2 | Sigma | M2320 | Mouse monoclonal |
| Anti-HIF1alpha | abcam | ab179483 | rabbit monoclonal |
| Anti-S100B | Sigma | HPA015768 | Rabbit polyclonal |
| Anti-GFAP (cocktail) | Biolegend | 837602 | |
| VECTASTAIN Elite ABC Kit (Rabbit IgG) | Vector Labs | PK-6101 | Contains 4 Reagents |
| Goat Anti Rabbit Alexa-Fluor 488 | Invitrogen | A11070 | |
| Goat Anti Mouse Alexa-Fluor 568 | Invitrogen | A11004 | |
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