NES and ischemia: To test this hypothesis, we cultured naïve (unchallenged) primary mouse astrocytes, astrocytes subjected to ischemia and astrocytes exposed to LPS in the presence or absence of C3a and used quantitative real time PCR (qRT-PCR) to determine the expression of genes coding for intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and nestin, C3aR, C3, nerve growth factor (NGF), tumor necrosis factor (TNF) and interleukin-1β (IL1β).