Establishment of a long-term spiral ganglion neuron culture with reduced glial cell number: Effects of AraC on cell composition and neurons
Establishment of a long-term spiral ganglion neuron culture with reduced glial cell number: Effects of AraC on cell composition and neurons
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DOI：
10.1016/j.jneumeth.2016.05.001
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发表时间：
2016-08-01
影响因子：
3
通讯作者：
Scheper, Verena
中科院分区：
文献类型：
--
作者：
Schwieger, Jana;Esser, Karl-Heinz;Scheper, Verena
背景：感音神经性耳聋主要由毛细胞损伤和螺旋神经节神经元变性引起。人工耳蜗术后可功能性替代毛细胞丢失，电刺激孤束核。植入人工耳蜗所带来的益处取决于这些神经元的数量和兴奋性。为了确定保护SGN的潜在治疗方法，对螺旋神经节细胞(SGC)进行了体外实验。新方法：提出了一种在有丝分裂抑制(阿糖胞苷，AraC)下培养新生大鼠SGC的神经胶质细胞减少和神经元增强培养长达7天的方法。结果：经AraC处理后，细胞总数明显减少，神经元比例明显增加。细胞类型特异性标记显示，几乎所有的成纤维细胞和大部分神经胶质细胞都被移除。对神经元存活、突起生长和胞体直径均无不良影响。与现有方法相比：目前的培养条件只允许短期培养新生SGC，缺乏关于非神经元细胞对SGN的影响以及神经突起与实验材料直接接触的信息。结论：Arac的加入减少了培养中非神经元细胞的数量，增加了SGN的比例，而对神经元的活性没有负面影响。这种处理方法允许长期培养SGC，并提供了对SGN-神经胶质细胞相互作用和突起在测试材料表面附着的更深层次的了解。(C)2016年提交人。爱思唯尔出版公司(Elsevier B.V.)
Background: Sensorineural deafness is mainly caused by damage to hair cells and degeneration of the spiral ganglion neurons (SGN). Cochlear implants can functionally replace lost hair cells and stimulate the SGN electrically. The benefit from cochlear implantation depends on the number and excitability of these neurons. To identify potential therapies for SGN protection, in vitro tests are carried out on spiral ganglion cells (SGC).New method: A glial cell-reduced and neuron-enhanced culture of neonatal rat SGC under mitotic inhibition (cytarabine (AraC)) for up to seven days is presented. Serum containing and neurotrophin-enriched cultures with and without AraC-addition were analyzed after 4 and 7 days.Results: The total number of cells was significantly reduced, while the proportion of neurons was greatly increased by AraC-treatment. Cell type-specific labeling demonstrated that nearly all fibroblasts and most of the glial cells were removed. Neither the neuronal survival, nor the neurite outgrowth or soma diameter were negatively affected. Additionally neurites remain partly free of surrounding non-neuronal cells.Comparison with existing method: Recent culture conditions allow only for short-term cultivation of neonatal SGC and lack information on the influence of non-neuronal cells on SGN and of direct contact of neurites with test-materials.Conclusions: AraC-addition reduces the number of non-neuronal cells and increases the ratio of SGN in culture, without negative impact on neuronal viability. This treatment allows longer-term cultivation of SGC and provides deeper insight into SGN-glial cell interaction and the attachment of neurites on test-material surfaces. (C) 2016 The Authors. Published by Elsevier B.V.