Chinese Medical E-ournals Database

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2016, Vol. 12 ›› Issue (03): 286 -290. doi: 10.3877/cma.j.issn.1673-5250.2016.03.007

Special Issue:

Original Article

Establishment of the model of oxygen-glucose deprivation and reperfusion in hippocampus neurons from neonatal rat in vitro

Hui Guo1, Dan Yu1, Hui Zhou1, Yu Tong1, Yuhong Tao1,()   

  1. 1. Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
  • Received:2016-03-22 Revised:2016-04-29 Published:2016-06-01
  • Corresponding author: Yuhong Tao
  • About author:
    Corresponding author: Tao Yuhong, Email:
Objective

To culture primary hippocampus neurons and establish a better oxygen-glucose deprivation and reperfusion (OGD/R) model for further research on neurons in vitro.

Methods

L-poly lysine and rat tail collagen were used as the growth medium in vitro culture of neurons, then hippocampal tissues were isolated from the newborn SD rats (within 24 hours of the birth), digested by single trypsin and bath shock, and made into cell suspension. The suspension was cultured with nutrient solution. 7 days later, to replace the medium with sugar free blood, and expose neurons to hypoxia in a three gas incubator containing 95% nitrogen (N2) for 2 hours, then cells were returned to former incubator under normoxic conditions for 24 hours to build a OGD/R model. Evaluating the OGD/R model with observing the morphological changes of neurons and the cell viability with anti-microtubule-associated protein-fluorescein-5-isothiocyanate (anti-MAP2-FITC) immunofluorescent staining and cell activity assay kit cell counting kit-8 (CCK-8) before and after the establishment of OGD/R model.

Results

With a fluorescence microscope, neuronal dendrites and axons stretched and overlapped into a network under normal circumstances were observed. After the establishment of OGD/R model, neuronal synapses retracted, cell disintegrated, reticular structure was destroyed and a decrease in neuronal viability was found by CCK8 test.

Conclusions

The neurons obtained by the aboved method were in good growth state, and the purity was higher than 95%. The OGD/R model of hippocampus neurons was successfully established by the way of replacing the normal medium with the sugar free medium combined with high purity N2 for 2 hours from neonatal rat in vitro.

图3 荧光显微镜下观察OGD/R建模对体外培养7 d的大鼠海马神经元细胞的影响(图3A:正常对照组;图3B:OGD/R组)(anti-MAP2-FITC免疫荧光染色,高倍镜)
表1 采用CCK-8测定OGD/R建模对海马神经元细胞生存率的影响
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