Optimal acting time of cytarabine in primary culture of rat cortical neurons

doi:10.3969/j.issn.2095-4344.2017.12.019

Abstract

Abstract:

BACKGROUND: Toxic cytarabine is often used to prepare highly purified neurons in experimental studies addressing central nervous system diseases. However, the intervention time of cytarabine is little reported.
OBJECTIVE: To determine the optimal intervention time of cytarabine(final concentration 10 μmol/L) in primary culture of rat cortical neurons.
METHODS: Rat primary cortical neurons were cultured in Neurobasal+B27 medium, and 10 μmol/L cytarabine were added at 12, 24, 36 and 48 hours after culture, respectively. Half of the medium were changed every 48 hours. The morphology of neurons was observed under inverted microscope at 7 days. The purity and differentiation of neurons maturity were identified by immunocytochemistry method of neuron specific enolization enzyme staining. Morphometric analysis for all neuron-specific enolase positive cells was performed by Multifunction Computer Image Analysis System.
RESULTS AND CONCLUSION: After addition of cytarabine at 24 hours of culture, the purity of neurons was more than 90%, well-differentiated cortical neurons accounted for 89.00%, and the area of neuronal body was the largest with the longest synapses. There were more neuron cells with transparent cytoplasm, and large nucleus. The cell body had good refraction and strong stereo sense. The neurons with 3-4 synapses and 3-4 bifurcates formed a good network structure. These results illustrate that although it will be beneficial for the purity of neurons to add cytarabine early in neuron culture process, it will make obvious effect on neuronal differentiation. The highly purified and well-grown cerebral cortical neurons will be obtained after cultured in neurolbasal medium, which cytarabine is added to at 24 hours of culture.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Cytarabine, Cerebral Cortex, Neurons, Cells, Cultured, Tissue Engineering

CLC Number:

R318

Guan Hong1, Pan Xue-feng2, 3, Liu Hao-kun4, Liu Xiao-qing1, Zhang Li-na2, Wang Shao-yi2, Dong Xiao-dong2, Niu Si-yun2. Optimal acting time of cytarabine in primary culture of rat cortical neurons[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(12): 1915-1920.

Figures/Tables 1

References

[1] 曾可斌,胡长林,陈阳美.大鼠海马神经元培养与鉴定[J].基础医学与临床,2004,24(5):574-578.

[2] 袁琼兰,郭勇,王琼,等.大鼠脑组织神经元培养?纯化?传代?鉴定[J].泸州医学院学报,2003,26 (3):200-205.

[3] 马万,袁文俊,焦英甫,等.一种稳定高效的新生大鼠皮质神经元原代培养方法[J].神经解剖学杂志,2015,31(4):510-514.

[4] 唐仕军,赵冬,朱立仓,等.一种简便的皮层神经元原代培养方法的建立及其培养结果分析[J].山东医药,2015,55(48):19-24.

[5] 曹珂,刘检,苗露阳,等.皮质神经元B27原代培养及MAP2鉴定[J].沈阳药科大学学报,2013,30(1):41-45.

[6] 赵康峰,顾雯,李玲,等大鼠海马神经元的原代培养及细胞鉴定[J]. 环境与健康杂志,2013,30(9):767-769.

[7] 李春莉,杨宝峰,张景海.新生大鼠海马和皮层神经元的原代培养及鉴定[J].沈阳药科大学学报,2011,28(4):300-304.

[8] Majd S, Zarifkar A, Rastegar K, et al.Culturing adult rat hippocampal neurons with long-interval changing media. Iran Biomed J. 2008;12(2):101-107.

[9] 李妙龄,杨艳,曾晓荣,等.新生大白鼠大脑皮层神经元的培养及其基本电生理学特性研究[J].泸州医学院学报, 2003,26(5): 378-381.

[10] Shimizu S, Abt A, Meucci O. Bilaminar co-culture of primary rat cortical neurons and glia. J Vis Exp. 2011;(57). pii: 3257.

[11] Gregory JB, John RT, Amanda LL,et al.Age-related toxicity of amyloid-beta associated with increased pERK and pCREB in primary hippocampal neurons: reversal by blueberry extract.J Nutr Biochem.2010;21(10):991–998.

[12] 李正伟,陈劲草,王媛,等.新生大鼠海马神经元原代培养方法改进[J].神经损伤与功能重建,2012, 7(2):88-90.

[13] 宋宁宁,宋丽,李剑,等.大鼠海马神经元元代培养方法及缺糖缺氧再关注损伤模型的建立[J].山东医药,2013,53(4):24-26.

[14] 周国凤, 汤京龙, 周亮,等.一种改进的大脑皮层神经元原代培养方法的研究药[J].物分析杂志,20 11, 31(2):299-310.

[15] 尹金宝,常全忠,韩宇东,等. 新生大鼠离体海马神经元原代培养方法及鉴定[J]. 神经解剖学杂志, 2012,28(6):613-616.

[16] 王廷华,冯忠堂.神经细胞培养理论与技术[M].北京:科学出版社2013:65−168.

[17] 黄立宁,韩建民,刘雅,等.新生大鼠海马神经元原代无血清培养与鉴定[J].基础医学与临床2012,32 (1):86-89.

[18] 杨传豪,赵冬,刘祺,等.新生大鼠皮层神经元体外无血清原代培养[J].重庆医学,2014,43(29):3901-3906.

[19] 康国创,刘文博,尹丽鹤,等.一种改进的高密度大鼠皮层神经元培养方法.中华神经外科疾病研究杂志[J].2009,8(5):417-420.

[20] Xiangmin Xu,Keith DR,Edward MC,et al.Immunochemical characterization of inhibitory mouse cortical neurons: Three chemically distinct classes of inhibitory cells.J Comp Neurol. 2010;518(3): 389-404.

[21] Kaneko A, Sankai Y. Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix.PLoS One.2014;9(7):e102703.

[22] 芦美玲,林霓阳,房晓祎.新生SD大鼠皮质神经元的体外培养和鉴定[J].生物技术通讯,2013, 24(3):402-405.

[23] Patel RS, Rachamalla M, Chary NR, et al.Cytarabine induced cerebellar neuronal damage in juvenile rat: correlating neurobehavioral performance with cellular and genetic alterations.Toxicology. 2012;293(1-3):41-52.

[24] 芦美玲,房晓祎,林霓阳.新生SD大鼠皮质神经元缺氧模型的改进[J].广东医学,2014,35(1): 53-55.

[25] 宋海岩,王志勇,李娜娜.新生大鼠大脑皮质神经元的原代培养及其鉴定[J].实用儿科临床杂志, 2012, 27(2);129-131.

[26] 辛岗,苏芸,王革非，等.新生BALB/c小鼠大脑皮质神经元细胞培养方法的建立[J].生物技术通讯, 2011, 22(1)：85-88.

[27] 周礼华,徐淑秀,江城梅.新生大鼠小脑颗粒神经元原代培养与鉴定[J].蚌埠医学院学报, 2011, 36(2):121-123.

[28] 张玮,唐卉凌,王筠,等.李澎涛大鼠大脑皮质神经元原代培养不同培养体系及纯化方法的比较研究[J].现代生物医学进展,2010, 10(6):1043-1046.

[29] 李昂,晏芳,李振林,等. 探讨阿糖胞苷在原代大鼠海马神经元培养中的纯化方法[J].中国临床解剖学杂志2015,33(6):681-684.

[30] Sui-Yi Xu, Yong-Min Wu, Zhong Ji, et al.A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons.J Biomed Biotechno.2012;(80):3930.