Primary Cultivation and Identification of Cortical Neuron in Neonatal Rats

Objective To establish the primary cultured method of cortical neuron in neonate rats for obtaining more neuron with high purity. Methods The cerebral cortex of SD rat with postnatal one day was received,and the monoplast suspension was obtained by enzyme digestion and then cultured in six orifices after calculated under inverted phase contrast microscope.After cultured 24 hours,the cells were treated by 10 μmol·L-1 cytarabine.The growth and morphologic change of cells were observed under inverted phase contrast microscope,the neurons were identified by Nissl′s staining,and the theneuronspecific enolase was displayed by immunofluorescence to indentify the purity of neuron. Results After culture,most neuron adhered with strongly three-dimensional round morphous at 2 hours,short dendrites appeared in most neuron and the morphous changed with round or oval appearance at 24 hours,the dendrites prolonged and thickened with many branch at 4 days,the neuron with well-stacked cell body and obvious halation formed the integrated neural network from 6 days to 8 days,then the neuron began to degenerate at 14 days.The Nissl body were hepatic particle and plaque stained by cresyl violet at 6 days after culture under inverted phase contrast microscope.The fluorescence staining showed that most neuron were stained in green with well morphous and clear nuclear,the rate of neuron in all cells was high. Conclusion The method used in this study is simple and economically for culturing cortical neuron,which can be accurately indentified through morphous observation,Nissl′s staining and fluorescence staining.