Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may pro-tect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotec-tive effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotro-phic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis. Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may pro-tectogenic neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotec-tive effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present protocols sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotropeic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.