Evaluation of the neurotoxic effects of ethanol on the cerebellar and cortical neurospheres isolated from E14 mouse embryo

Abstract

Objectives: Ethanol exposure during pregnancy has toxic effects on the neural stem cells of the developing brain. Differential effects of the alcohol on the neural precursor cells from different regions of the brain have not been established. This study was planned to find out the difference in the morphology of the neurosphere (NS) from the embryonic mouse cortex and cerebellum when exposed to ethanol. The dose of ethanol was comparable to blood alcohol concentration in binge drinking. Materials and methods: The germinal tissues around the lateral ventricle and fourth ventricle of the embryo of pregnant albino mice (E14) were dissected out. Primary cultures were plated using adequate density of cells in media hormone mix (MHM) and growth factors. After 48 hours, ethanol was added to the cultures (low dose = 80 mg/dl; high dose = 400 mg/dl). NS from different groups were analyzed using phase-contrast microscopy, Trypan blue exclusion assay and immunostaining with 4'-6-Diamidino-2-phenylindol (DAPI). Results: With low dose ethanol treatment, the mean area of cortical neurospheres was reduced by 6.9% whereas that of the cerebellar neurospheres was reduced by 51.2%. The mean count of pyknotic cells increased significantly (p<0.05) in both low and high dose ethanol treatment group compared to control in both cortical and cerebellar culture. Cerebellar culture showed 4-fold-increase in mean pyknotic cells compared to 2.5-fold-increase in cortical culture, when exposed to ethanol. Conclusions: Cerebellar neural precursor cells from E14 mouse were more susceptible to the neurotoxic effects of ethanol compared to the cortical ones.