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Primary Neuron-glia Culture from Rat Cortex as a Model to Study Neuroinflammation in CNS Injuries or Diseases
大鼠皮质原代神经元和胶质细胞共培养作为中枢神经系统神经炎症的研究模型

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Abstract

Primary neuron-glia cultures are commonly used in vitro model for neurobiological studies. Here, we provide a protocol for the isolation and culture of neuron-glial cells from cortical tissues of 1-day-old neonatal Sprague-Dawley pups. The procedure makes available an easier way to obtain the neuron and glia. In this culture system, neuron-glia cultures consisted of approximately 37% neurons, 51% astrocytes, 7% microglia, and a small percentage (<5%) of other cells after fourteen days in vitro. Primary neuron-glia cultures is a simplified in vitro model for studies focusing on interactions between neurons and glia cells. Activated glial cells, mainly astrocytes and microglia, are histopathological hallmarks of acute injury of the central nervous system (CNS) or chronic neurologic diseases (Hirsch and Hunot, 2009; Lee et al., 2009; Minghetti, 2005). Inflammatory mediators (e.g. nitric oxide, reactive oxygen species, proinflammatory cytokines, and chemokines) released by activated glia can directly or indirectly cause neuronal damage or neurodegeneration. Neuroinflammation is a common mechanism of various neurological diseases leading to neurodegeneration. The advantages of neuron-glia cultures are that: (1) Cultured cells can bypass complicated physiological interactions (such as leukocyte infiltration, blood-brain barrier, reflex or other systemic regulation) in vivo to allow direct observation of neuroinflammation caused by various CNS insults (hypoxia, ischemia, trauma. infection, neurotoxins, chronic stress or diseases); (2) Unlike cell lines that are mostly derived from tumor cells, primary cultured neuron-glia system is closer to the cell population ratio in vivo and can mimic the in situ microenvironment; and (3) Cultures can be prepared from various brain regions (e.g. cortex, hippocampus, mesencephalon…etc) and allow an opportunity to examine the regional difference in the susceptibility to neurodegeneration following neuroinflammation caused by various CNS insults (Kim et al., 2000). The following protocol is an example for primary rat cortical neuron-glia culture preparation (Huang et al., 2015; Huang et al., 2014; Huang et al., 2012; Huang et al., 2009).

Keywords: Primary neuron-glia culture(原发性神经胶质细胞培养), Neuroinflammation(神经炎症), CNS injuries(中枢神经系统损伤), I(我)

Materials and Reagents

  1. Tissue culture dishes (60 x 15 mm) (Sigma-Aldrich, catalog number: P5237 )
  2. Tissue culture dishes (100 x 20 mm) (Nunc, catalog number: 172958 )
  3. Screw cap centrifuge tube (50 ml) (Sigma-Aldrich, catalog number: BR114821 )
  4. 24-well plates (Sigma-Aldrich, catalog number: CLS 3527 )
  5. Pipette tips (10 μl, 200 μl and 1,000 μl) (Shineteh instruments co ltd., catalog number: PT4-W10 , PT1-Y200 and PT5-W10 )
  6. Neonatal Sprague-Dawley pups (1-day-old)
  7. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  8. Hanks’ Balanced Salt solution (HBSS) (Sigma-Aldrich, catalog number: 55021C )
  9. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  10. Pyruvate (Sigma-Aldrich, catalog number: P2256 )
  11. HEPES (Sigma-Aldrich, catalog number: H3375 )
  12. Bovine Serum Albumins (BSA) (Sigma-Aldrich, catalog number: A9418 )
  13. DMED powder (Thermo Fisher Scientific, GibcoTM, catalog number: 12100-046 )
  14. DMED/F-12, HEPES, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 11039-021 )
  15. Penicilline/Stretomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  16. 100 mM Sodium pyruvate solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070 )
  17. MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050 )
  18. Fetal bovine serum (FBS) (NQBB, catalog number: A6806-11 )
  19. Hank’s solution (see Recipes)
  20. Dulbecco's modified Eagle’s medium (DMEM) (see Recipes)
  21. Serum-free medium (100 ml) (see Recipes)

Equipment

  1. 37 °C, 5% CO2 incubator (Water Jacketed Laboratory CO2 Incubator)
  2. Stereo microscope (Shineteh instruments co., catalog number: IH1-ZM150A )
  3. Biological Inverted microscope (OLYMPUS CORPORATION, model: IX71 )
  4. Mini micro centrifuge (Shineteh instruments co., catalog number: IC-MINIMAX )
  5. Centrifuge (Hermle, catalog number: Hermle Z232K )
  6. Water bath (Bioman Scientific Co ltd, catalog number: SWB-20-1 )
  7. Counting chamber (Shineteh instruments co., catalog number: PT14-901001 )
  8. Operating scissors STR (Shineteh instruments co., catalog number: ST-014 )
  9. Iris scissors STR (Shineteh instruments co., catalog number: ST-S009 )
  10. Dressing forceps (Shineteh instruments co., catalog number: ST-D114 )
  11. Iris forceps (Shineteh instruments co., catalog number: ST-I510 )
  12. Tweezers forceps (Shineteh instruments co ltd., catalog number: ST-NO5 )
  13. Pipetman (Gilson, catalog number: P10 , P200 and P1000)
  14. Pipet-aid (Thomas Scientific, FlaconTM, catalog number: 0410C04 )
  15. Pipets (10 ml) (Tseng Hsiang Life Science ltd., catalog number: SP-1-C )
  16. Ice bucket (Shineteh instruments co ltd., catalog number: PA8-4 )

Procedure

  1. Primary neuron-glia cultures are prepared from cortical tissues of 1-day-old neonatal Sprague-Dawley pups.
  2. After the rats are sacrificed, their brains are quickly removed aseptically. Under the stereo surgical microscope, the blood vessels and meninges are removed by tweezers.
  3. Cerebral cortices (6~8 pups cerebral cortices /tube) are dissected under sterile conditions and kept on ice in conical tube containing 10 ml of Hank’s solution (without Ca2+ or Mg2+).
  4. Remove Hank’s solution and add 15 ml of warm (37 °C) Dulbecco's modified Eagle’s media (DMEM) containing 10% heat-inactivated FBS to 50 ml conical tube.
  5. Cortical cells are dissociated by trituration using a 10 ml pipette (about 15 times). Cells are centrifuged at 4 °C (1,500 x g) for 5 min to pellet cells.
  6. After centrifuged, cells are re-suspended in 10 ml of DMEM containing 10% heat-inactivated FBS.
  7. Dilute cells for counting by adding 20 μl of cells to 180 μl of culture medium (10x dilution).
  8. Add 10 μl of the above cell dilution to a hemocytometer. Cells are counted in the 1 mm center square.
  9. Cell density is determined by the average of the 5 randomly selected squares. To calculate the total # of cells/ml = (the average count per square) x (the dilution factor) x (105/ml).
  10. Dilute the 10 ml stock to make a final concentration of 5 x 105 cells/ml.
  11. To each well of 24-well culture plates 5 x 105 cells are seeded in 0.5 ml of culture medium.
  12. The cultures are incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
  13. Culture medium is replenished five days after plating, and is changed every three days thereafter.
  14. The neuron-glial cells become confluent 10-12 days after plating.
  15. Fourteen days in vitro (DIV14), the cultures are used for experiment. The cell composition is determined by immunostaining, followed by cell counting. The neuron-glia cultures consisted of approximately 37% ± 0.8% neurons, 51% ± 1.5% astrocytes, and 7% ± 7.7% microglia (Figure 1). In addition, cultures also consist of a small percentage (<5%) of other cells including oligodendrocytes, fibroblast and endothelial cells (data not shown).
  16. The characterization of neuronal phenotypes is shown in Figure 2.


    Figure 1. The composition of cell types in primary cortical neuron-glia cultures. Phase contrast photomicrograph of the primary cortical neuron-glia cultures in vitro (DIV14) (a), and representative immunocytochemically-stained photomicrographs using antibodies against (neuronal) dendritic marker microtubule associated protein 2 (MAP2) (b), astrocytic marker glial fibrillary acidic protein (GFAP) (c) or pan-macrophage lysosomal antigen (ED1) as a phagocytic marker for activated microglia (d). Percentage of immunostain positive cells of neurons (MAP2+), astrocytes (GFAP+) and activated microglia (ED1+) cells in cultures (e). Scale bar = 25 μm


    Figure 2. Characterization of neuronal phenotype by immunocytochemical staining. Cultures were stained using antibodies against (a) neuronal marker neuronal nuclei antigen (NeuN) on 14 days in vitro (DIV 14), (b) excitatory amino acid carrier 1 (EAAC1) for glutamatergic neurons, (c) 5-hydroxytryptamine (5HT) for serotonergic neurons, (d) glutamate decarboxylase 65 (GAD 65) for GABAergic neurons, (e) choline acetyltransferase (ChAT) for cholinergic neurons, and (f) tyrosine hydroxylase (TH) for catecholaminergic neurons. Scale bar = 25 μm

Recipes

  1. Hank’s solution (1,000 ml)
    Reagents
    Volume
    Hanks’ balanced salt solution
    1,000 ml
    Sodium bicarbonate
    0.35 g
    Pyruvate
    0.11 g
    HEPES
    4.76 g
    Bovine serum albumins (BSA)
    3 g
  2. 10% fetal bovine serum (FBS) / Dulbecco's modified Eagle’s medium (DMEM) (1,000 ml)
    Reagents
    Volume
    DMED powder
    1 package
    Sodium bicarbonate
    2.2 g
    Pyruvate
    0.11 g
    HEPES
    4.76 g
    FBS
    110 ml
  3. Serum-free medium (100 ml) (drug treatment only)
    Reagents
    Volume
    DMED/F-12, HEPES, no phenol red
    97 ml
    100x Penicilline/Stretomycin
    1 ml
    100x sodium pyruvate solution (100 mM)
    1 ml
    100x MEM non-essential amino acids solution
    1 ml

Acknowledgments

This protocol is adapted from the previously published studies (Huang et al., 2015; Huang et al., 2014; Huang et al., 2009; Kim et al., 2000). These studies were supported in part by grants from the Hsin Sheng Junior College of Medical Care and Management (HSC-103-001 and HSC-104-015) and from Taipei Medical University (03C0720004A) to YNH; and from the Ministry of Science and Technology (MOST-104-2923-B-038-001-MY3 and MOST-104-2320-B-038-057-MY3) to JYW, Taiwan.

References

  1. Hirsch, E. C. and Hunot, S. (2009). Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol 8(4): 382-397.
  2. Huang, Y. N., Ho, Y. J., Lai, C. C., Chiu, C. T. and Wang, J. Y. (2015). 1,25-Dihydroxyvitamin D3 attenuates endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation in primary cortical neuron-glia cultures. J Neuroinflammation 12: 147.
  3. Huang, Y. N., Lai, C. C., Chiu, C. T., Lin, J. J. and Wang, J. Y. (2014). L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF-kappaB translocation in cortical neurons/glia Cocultures. PLoS One 9(7): e97276.
  4. Huang, Y. N., Wang, J. Y., Lee, C. T., Lin, C. H., Lai, C. C. and Wang, J. Y. (2012). L-Ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1. Toxicology and Applied Pharmacology 265(2): 241-252.
  5. Huang, Y. N., Wu, C. H., Lin, T. C. and Wang, J. Y. (2009). Methamphetamine induces heme oxygenase-1 expression in cortical neurons and glia to prevent its toxicity. Toxicology and Applied Pharmacology 240(3): 315-326.
  6. Kim, W. G., Mohney, R. P., Wilson, B., Jeohn, G. H., Liu, B. and Hong, J. S. (2000). Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J Neurosci 20(16): 6309-6316.
  7. Lee, Y., Lee, S. R., Choi, S. S., Yeo, H. G., Chang, K. T. and Lee, H. J. (2014). Therapeutically targeting neuroinflammation and microglia after acute ischemic stroke. Biomed Res Int 2014: 297241.
  8. Minghetti, L. (2005). Role of inflammation in neurodegenerative diseases. Curr Opin Neurol 18(3): 315-321.

简介

原代神经元 - 神经胶质培养物通常用于神经生物学研究的体外模型。在这里,我们提供的隔离和培养的神经元胶质细胞从1日龄新生儿Sprague-Dawley幼崽的皮质组织的协议。该程序提供了一种更容易的方式来获得神经元和神经胶质。在该培养系统中,神经元 - 神经胶质培养物在体外14天后由约37%神经元,51%星形胶质细胞,7%小胶质细胞和小百分比(<5%)的其他细胞组成。原代神经元胶质细胞培养物是用于研究的简化的体外模型,其关注于神经元和神经胶质细胞之间的相互作用。活化的胶质细胞,主要是星形胶质细胞和小神经胶质细胞,是中枢神经系统(CNS)或慢性神经疾病的急性损伤的组织病理学标志(Hirsch和Hunot,2009; Lee等人,2009; Minghetti, 2005)。由活化神经胶质释放的炎性介质(例如一氧化氮,活性氧,促炎细胞因子和趋化因子)可直接或间接引起神经元损伤或神经变性。神经炎症是导致神经变性的各种神经疾病的常见机制。神经胶质细胞培养的优点是:(1)培养的细胞可以在体内绕过复杂的生理相互作用(例如白细胞浸润,血脑屏障,反射或其他系统调节)以允许直接观察(缺氧,缺血,创伤,感染,神经毒素,慢性应激或疾病)引起的神经炎症; (2)与主要源自肿瘤细胞的细胞系不同,原代培养的神经元 - 神经胶质系统更接近于体内细胞群体比率,并且可以模拟原位微环境;和(3)可以从各种脑区域(例如皮质,海马,中脑等)制备培养物,并且允许有机会检查对神经变性的易感性的区域差异随后由各种CNS损伤引起的神经炎症(Kim等人,2000)。以下方案是原代大鼠皮质神经元 - 神经胶质培养物制备的实例(Huang等人,2015; Huang等人,2014; Huang等人。,2012; Huang ,,2009)。

关键字:原发性神经胶质细胞培养, 神经炎症, 中枢神经系统损伤, 我

材料和试剂

  1. 组织培养皿(60×15mm)(Sigma-Aldrich,目录号:P5237)
  2. 组织培养皿(100×20mm)(Nunc,目录号:172958)
  3. 螺旋盖离心管(50ml)(Sigma-Aldrich,目录号:BR114821)
  4. 24孔板(Sigma-Aldrich,目录号:CLS3527)
  5. 移液管吸头(10μl,200μl和1000μl)(Shineteh instruments co ltd。,目录号:PT4-W10,PT1-Y200和PT5-W10)
  6. 新生的Sprague-Dawley幼仔(1日龄)
  7. 台盼蓝(Thermo Fisher Scientific,GibcoTM,目录号:15250061)
  8. Hanks平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:55021C)
  9. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  10. 丙酮酸(Sigma-Aldrich,目录号:P2256)
  11. HEPES(Sigma-Aldrich,目录号:H3375)
  12. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  13. DMED粉末(Thermo Fisher Scientific,GibcoTM,目录号:12100-046)
  14. DMED/F-12,HEPES,无酚红(Thermo Fisher Scientific,GibcoTM,目录号:11039-021)
  15. 青霉素/链霉素(100x)(Thermo Fisher Scientific,GibcoTM,目录号:15140-122)
  16. 100mM丙酮酸钠溶液(100x)(Thermo Fisher Scientific,GibcoTM,目录号:11360-070)
  17. MEM非必需氨基酸溶液(100x)(Thermo Fisher Scientific,GibcoTM,目录号:11140-050)
  18. 胎牛血清(FBS)(NQBB,目录号:A6806-11)
  19. 汉克的解决方案(见配方)
  20. Dulbecco改良的Eagle培养基(DMEM)(参见Recipes)
  21. 无血清培养基(100ml)(见配方)

设备

  1. 37℃,5%CO 2培养箱(水夹套实验室CO 2培养箱)中。
  2. 立体显微镜(Shineteh instruments co。,目录号:IH1-ZM150A)
  3. 生物反转显微镜(OLYMPUS CORPORATION,型号:IX71)
  4. 微型离心机(Shineteh instruments co。,目录号:IC-MINIMAX)
  5. 离心机(Hermle,目录号:Hermle Z232K)
  6. 水浴(Bioman Scientific Co ltd,目录号:SWB-20-1)
  7. 计数室(Shineteh instruments co。,目录号:PT14-901001)
  8. 操作剪STR(Shineteh instruments co。,目录号:ST-014)
  9. 虹膜剪STR(Shineteh instruments co。,目录号:ST-S009)
  10. 敷料钳(Shineteh instruments co。,目录号:ST-D114)
  11. Iris镊子(Shineteh instruments co。,目录号:ST-I510)
  12. 镊子钳(Shineteh instruments co ltd。,目录号:ST-NO5)
  13. Pipetman(Gilson,目录号:P10,P200和P1000)
  14. 移液管(Thomas Scientific,FlaconTM,目录号:0410C04)
  15. 吸管(10ml)(Tseng Hsiang Life Science有限公司,目录号:SP-1-C)
  16. 冰桶(Shineteh instruments co ltd。,目录号:PA8-4)

程序

  1. 原代神经元胶质细胞培养物从1日龄新生儿Sprague-Dawley幼鼠的皮质组织制备。
  2. 在处死大鼠后,无菌地快速除去其大脑。在立体外科显微镜下,用镊子除去血管和脑膜
  3. 在无菌条件下解剖大脑皮质(6?8只大脑皮层/管),并保持在冰上在含有10ml Hank's溶液(不含Ca 2+或Mg 2+)/sup>)。
  4. 取出Hank's溶液,并加入15ml含有10%热灭活的FBS的温热(37℃)Dulbecco改良的Eagle培养基(DMEM)至50ml锥形管。
  5. 使用10ml移液管通过研磨使皮层细胞解离(约15次)。将细胞在4℃(1,500xg)离心5分钟以沉淀细胞。
  6. 离心后,将细胞重悬于10ml含有10%热灭活的FBS的DMEM中。
  7. 通过将20μl细胞加入到180μl培养基(10倍稀释)中稀释细胞用于计数。
  8. 加入10μl的上述细胞稀释液到血细胞计数器。细胞在1mm中心正方形中计数。
  9. 细胞密度由5个随机选择的正方形的平均值确定。为了计算总细胞数/ml =(每平方的平均计数)×(稀释因子)×(10 /ml)。
  10. 稀释10ml原液,使最终浓度为5×10 5细胞/ml。
  11. 向24孔培养板的每个孔中,将5×10 5个细胞接种在0.5ml培养基中。
  12. 将培养物在37℃下在5%CO 2和95%空气的湿润气氛中孵育。
  13. 培养基在铺板后5天补充,然后每三天更换一次
  14. 电镀后10-12天神经元胶质细胞变成融合
  15. 体外14天(DIV14),将培养物用于实验。通过免疫染色,然后通过细胞计数测定细胞组成。神经胶质细胞培养物由约37%±0.8%神经元,51%±1.5%星形胶质细胞和7%±7.7%小胶质细胞组成(图1)。此外,培养物还包括少量(<5%)的其他细胞,包括少突胶质细胞,成纤维细胞和内皮细胞(数据未显示)。
  16. 神经元表型的表征如图2所示

    图1.原代皮层神经元胶质细胞培养物中细胞类型的组成。原代皮质神经胶质细胞培养物的体外显微镜照片(DIV14)(a),和使用抗(神经元)树突标记微管相关蛋白2(MAP2)(b),星形细胞标志物胶质纤维酸性蛋白(GFAP)(c)或泛巨噬细胞溶酶体抗原(ED1)作为吞噬标记的抗体的代表性免疫细胞化学染色的显微照片对于活化的小胶质细胞(d)。培养物中神经元(MAP2 + +),星形胶质细胞(GFAP +)和活化的小胶质细胞(ED1 +)的免疫染色阳性细胞的百分比)。比例尺=25μm


    图2.通过免疫细胞化学染色表征神经元表型。使用针对(a)神经元标记神经元核抗原(NeuN)的抗体在14天体外染色培养物(DIV 14 ),(b)谷氨酸能神经元的兴奋性氨基酸载体1(EAAC1),(c)5-羟色胺能神经元的5-羟色胺(5HT),(d)GABA能神经元的谷氨酸脱羧酶65(GAD 65),(e)胆碱乙酰转移酶ChAT)和(f)儿茶酚胺能神经元的酪氨酸羟化酶(TH)。比例尺=25μm

食谱

  1. 汉克氏溶液(1000ml)
    试剂

    汉克斯平衡盐溶液
    1000 ml
    碳酸氢钠
    0.35克
    丙酮酸
    0.11克
    HEPES
    4.76克
    牛血清白蛋白(BSA)
    3克
  2. 10%胎牛血清(FBS)/Dulbecco改良的Eagle培养基(DMEM)(1000ml)中培养。
    试剂

    DMED粉末
    1包
    碳酸氢钠
    2.2 g
    丙酮酸
    0.11克
    HEPES
    4.76克
    FBS
    110 ml
  3. 无血清培养基(100 ml)(仅药物治疗)
    试剂

    DMED/F-12,HEPES,无酚红
    97 ml
    100x青霉素/链霉素 1 ml
    100x丙酮酸钠溶液(100mM) 1 ml
    100x MEM非必需氨基酸溶液
    1 ml

致谢

该协议改编自先前公开的研究(Huang等人,2015; Huang等人,2014; Huang等人, 2009; Kim 等人,2000)。这些研究部分来自新生医学保健和管理学院(HSC-103-001和HSC-104-015)和台北医科大学(03C0720004A)到YNH的资助;和来自科技部(MOST-104-2923-B-038-001-MY3和MOST-104-2320-B-038-057-MY3)至台湾的JYW。

参考文献

  1. Hirsch,E.C。和Hunot,S。(2009)。 帕金森病中的神经炎症:神经保护的靶点 Lancet Neurol < em> 8(4):382-397。
  2. Huang,Y. N.,Ho,Y. J.,Lai,C. C.,Chiu,C.T.and Wang,J.Y。(2015)。 1,25-二羟基维生素D3通过抑制原代皮层中的MAPK激活而减弱内毒素诱导的炎症介质的产生神经元胶质细胞培养物。 12:147
  3. Huang,Y. N.,Lai,C. C.,Chiu,C. T.,Lin,J. J.和Wang,J. Y.(2014)。 L-抗坏血酸通过抑制MAPK激活和NF-kappaB易位减弱内毒素诱导的炎症介质的产生in cortical neurons/glia Cocultures。 PLoS One 9(7):e97276。
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引用:Huang, Y. and Wang, J. (2016). Primary Neuron-glia Culture from Rat Cortex as a Model to Study Neuroinflammation in CNS Injuries or Diseases. Bio-protocol 6(8): e1788. DOI: 10.21769/BioProtoc.1788.
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