RESEARCH ARTICLE Non-Neuronal Cells Are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System

Chronic inflammation is associated with activated microglia and reactive astrocytes and plays an important role in the pathogenesis of neurodegenerative diseases such as Alzhei-mer’s. Both in vivo and in vitro studies have demonstrated that inflammatory cytokine responses to immune challenges contribute to neuronal death during neurodegeneration. In order to investigate the role of glial cells in this phenomenon, we developed a modified method to remove the non-neuronal cells in primary cultures of E16.5 mouse cortex. We modified previously reported methods as we found that a brief treatment with the thymidine analog, 5-fluorodeoxyuridine (FdU), is sufficient to substantially deplete dividing non-neuro-nal cells in primary cultures. Cell cycle and glial markers confirm the loss of ~99 % of all microglia, astrocytes and oligodendrocyte precursor cells (OPCs). More importantly, under this milder treatment, the neurons suffered neither cell loss nor any morphological defects up to 2.5 weeks later; both pre- and post-synaptic markers were retained. Further, neurons in FdU-treated cultures remained responsive to excitotoxicity induced by glutamate applica-tion. The immunobiology of the FdU culture, however, was significantly changed. Compared with mixed culture, the protein levels of NFκB p65 and the gene expression of several cyto-kine receptors were altered. Individual cytokines or conditioned medium from β-amyloid-stimulated THP-1 cells that were, potent neurotoxins in normal, mixed cultures, were virtu-ally inactive in the absence of glial cells. The results highlight the importance of our glial-depleted culture system and identifies and offer unexpected insights into the complexity of-brain neuroinflammation