ABSTRACT
Objective To establish a stable primary culture of rat cerebellar granule neurons in vitro for further study the toxic effects of chronic arsenic exposure on cerebellar cells.Methods Cerebellar cortices were taken from brain of Wistar rat 5-7 day old after born under stereoscopic microscope.Single cell suspension was acquired after digestion and washing with trypsin (0.25％) and DNase Ⅰ solution,respectively.Granule cells were purified from other cells by differential velocity adherence method for two times.Rat cerebellar granule neurons were seeded in culture plate pre-coated with poly-L-lysine.Neurons growth,development and synaptic connections were observed daily.The neurons were identified by neuron specific enolase (NSE) immunofluorescence technique.Results The neurons were affixed to the culture plate in 24 hours,in reticular arrangement observed under contrast microscope.Granule cells gradually turned round from oval and outlines became clearer in 2-3 days.In 4-6 days,there were a wide range of synaptic connections among the neurons and a mature nerve cell network formed.A large quantity of cerebellar granule neurons was seen by NSE identification.Few bigger cells such as purkinjes cells and glial cell outlines were also seen in the same visual field.Conclusions This is a successful primary culture method for acquirement of rat cerebellar granule neurons.The method can provide experimental basis for future studies the toxic effects of chronic arsenic exposure on cerebellar cells.
ABSTRACT
Aim: To investigate the effects of insulin like growth factor1 (IGF-1) on apoptosis of cerebellar granule neurons (CGNs) induced by diphenylhydantoin (DPH) and its possible relationship with PI3K/Akt. Methods: Rat cerebellar granule neurons (CGNs), primarily cultured for 8 days, were co-incubated with 100 μmol·L-1 DPH and 1 μmol·L-1 IGF-1 for 48 h and then submitted to apoptotic analysis. CGNs, pretreated with 100 μmol·L-1 DPH and 1 μmol·L-1 IGF-1 for 48 h, were incubated with LY294002, a specific inhibitor of PI3K for 30 minutes, and then performed cell viability assays to explore the relationship of IGF-1 with PI3K/Akt pathway. Western blotting was employed to further study whether Akt was involved in the apoptotic effect of DPH and the protection of IGF-1. Results 1 μmol·L-1 IGF-1 demonstrated significant protective effects on apoptosis of CGNs treated with DPH, which could be abolished by LY294002, a specific inhibitor of PI3K/Akt pathway. IGF-1 also upregulated the activity of Akt in CGNs, which was markedly decreased by DPH. Conclusions: IGF-1 blocked the DPH-induced apoptosis of CGNs possibly through a PI3K/Akt dependent pathway.