Abstract
It has been demonstrated that in vivo brain ischemia induces activation and proliferation of astrocytes and microglia. However, the mechanism underlying the ischemia-induced activation and proliferation of these cells remains to be unclear. Oxygen-glucose deprivation (OGD), an in vitro ischemia mimic, has been extensively used to analyze the hypoxia response of various cell types. This study examined the OGD-induced changes in the expression level of astrocytes and microglia marker proteins and immunoreactivity for Ki-67, a marker protein for cell proliferation, using rat primary hippocampal neuron-glia co-culture (NGC) cells. Furthermore, OGD-induced changes in the expression of M1/M2 microglia phenotype-related genes were also examined. MTT assay indicated that 120 min of OGD decreased cell viability, and immunocytochemistry indicated that 120 min of OGD abolished most microtubule-associated protein 2 (MAP2)-immunopositive neurons. In contrast, glial fibrillary acidic protein (GFAP)-immunopositive astrocytes and ionized calcium-binding adapter protein-1 (Iba-1)-immunopositive microglia, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase)-immunopositive oligodendrocytes survived OGD. Western blot assays and double-immunofluorescent staining indicated that OGD increased the GFAP expression level and the Ki-67-immunopositive/GFAP-immunopositive cells' ratio. Real-time PCR analysis showed that OGD altered M1 microglia phenotype-related genes. Specifically, OGD decreased the expression level of CD32 and interleukin-1β (IL-1β) genes and increased that of the inducible nitric oxide synthase (iNOS) gene. Therefore, applying OGD to NGC cells could serve as a useful in vitro tool to elucidate the molecular mechanisms underlying brain ischemia-induced changes in GFAP expression, astrocyte proliferation, and M1 microglia phenotype-related gene expression.