Primary culture and identification of neurons from cerebral cortex of newborn Sprague-Dawley rats

doi:10.12307/2023.390

Abstract

Abstract: BACKGROUND: The quality of cultured neurons is the key to the study of central nervous system diseases, but there has been no unified standard for the methods used.

OBJECTIVE: To explore the effects of best selection site and cytarabine treatment conditions on the viability, purity and maturity of primary cultured neurons.

METHODS: The cells were obtained at 2.0-3.0 mm on the surface of the cerebral cortex of Sprague-Dawley rats within 24 hours of the newborn, and treated with cytarabine at a concentration of 5 μmol/L or 10 μmol/L at 24, 48, and 72 hours after culture. In addition, the whole cerebral cortex was removed for primary culture and treated with cytarabine at a concentration of 5 μmol/L for 48 hours at 48 hours after culture. At 7 days after culture, the morphology of neuron was observed using inverted microscope and the effect of cytarabine on neuronal viability was detected by CCK-8 assay. The purity and maturity of neurons were assessed by immunofluorescence and western blot assay.

RESULTS AND CONCLUSION: (1) Inverted microscope: The density of neurons in the 10 μmol/L cytarabine group at 24, 48, and 72 hours was lower than that in the 5 μmol/L cytarabine group at the same time point, and the neuronal processes in the six groups could be connected to each other to form a dense network structure. The neuron cell body was plump and full of refractive properties. The cell density of the whole cortex group was lower than that of the normal control group, and non-neuronal cells could be clearly observed. (2) CCK-8 assay: The cell viability of each cytarabine group was lower than that of the normal control group (P < 0.001), and the neuronal viability of the 10 μmol/L cytarabine 24-, 48-, and 72-hour groups was lower than that of 5 μmol/L cytarabine group at the same time point (P < 0.05). The neuronal viability in the 5 μmol/L cytarabine 24-hour group was lower than that in the 5 μmol/L cytarabine 48- and 72-hour group (P < 0.01). (3) Immunofluorescence staining: The purity of neurons in each cytarabine group was greater than that in the normal control group and all cortical groups (P < 0.001), and there was no difference in the purity of neurons in each cytarabine group. (4) Western blot assay detection of neuron-specific enolase protein expression: The neuron maturity of each cytarabine group was higher than that of the normal control group (P < 0.05), and the 5 μmol/L cytarabine 48-hour group had the highest maturity. (5) The results showed that the cortex at 2.0-3.0 mm on the surface of the rat cerebral cortex within 24 hours of the newborn treated with 5 μmol/L cytarabine at 48 hours after culture can obtain better neuronal viability, maturity and purity.

Key words: cytarabine, cerebral cortex, neuron, primary culture, neuronal viability, rat

CLC Number:

Wang Jing, Lu Sitong, Wu Yinlin, Liu Yudong, Zou Weiyan, Sun Meiqun. Primary culture and identification of neurons from cerebral cortex of newborn Sprague-Dawley rats[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(15): 2344-2349.

Figures/Tables 5

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