Minocycline, a tetracycline antibiotic, is now known to protect cells via an anti-inflammatory mechanism. We further explored this effect using an in vitro model of ischemia-like injury to neurons. Coculturing neurons with microglia, the brain's resident immune cell, modestly increased cell death due to oxygen and glucose deprivation (OGD), compared to neurons alone. Treatment of cocultures with minocycline decreased cell death to a level significantly lower than that of neurons alone. Treatment of cocultures with minocycline or inhibitors of various immune mediators, also led to decreased cell death. Importantly, treatment of neuron cultures without added microglia with these same inhibitors of tissue plasminogen activator, matrix metalloproteinases, TNF-alpha and inducible nitric oxide synthase as well as minocycline also led to decreased cell death. Thus, anti-inflammatory treatments appear to be directly protective of neurons from in vitro ischemia.

Abstract Background Activation of the NF-κB transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. Its activation is dynamic and is part of a network of biochemical species with multiple feedback regulatory mechanisms. Mathematical modeling, which has been instrumental for understanding the NF-κB response in other cell types, offers a valuable tool to investigate the regulation of NF-κB activation in microglia at a systems level. Results We quantify the dynamic response of NF-κB activation and activation of the upstream kinase IKK using ELISA measurements of a microglial cell line following treatment with the pro-inflammatory cytokine TNFα. A new mathematical model is developed based on these data sets using a modular procedure that exploits the feedback structure of the network. We show that the new model requires previously unmodeled dynamics involved in the stimulus-induced degradation of the inhibitor IκBα in order to properly describe microglial NF-κB activation in a statistically consistent manner. This suggests a more prominent role for the ubiquitin-proteasome system in regulating the activation of NF-κB to inflammatory stimuli. We also find that the introduction of nonlinearities in the kinetics of IKK activation and inactivation is essential for proper characterization of transient IKK activity and corresponds to known biological mechanisms. Numerical analyses of the model highlight key regulators of the microglial NF-κB response, as well as those governing IKK activation. Results illustrate the dynamic regulatory mechanisms and the robust yet fragile nature of the negative feedback regulated network. Conclusions We have developed a new mathematical model that incorporates previously unmodeled dynamics to characterize the dynamic response of the NF-κB signaling network in microglia. This model is the first of its kind for microglia and provides a tool for the quantitative, systems level study the dynamic cellular response to inflammatory stimuli.