Iron Activation of Cellular Oxidases: Modulation of Neuronal Viability (In Vitro)
Technical Report,01 Aug 2015,31 Aug 2017
Tri Service Nursing Research Program Rockville United States
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Purpose Traumatic brain injury TBI is a significant health issue with 361,092 service members diagnosed. Increased iron, oxidative stress and microglia are present within TBI lesions acutely after injury. Microglia are major modulators of the inflammatory response and produce neurotoxic reactive oxygen species ROS via NADPH oxidases NOX. We suspected that iron is a major contributor to neuro-degeneration by contributing to oxidative stress through microglia. We hypothesized that iron utilized NOX derived ROS to accentuate ROS synthesis inactivated microglia and reduced neuronal survivability in vitro. Design Experimental design that utilized immortalized BV2 microglia, 8 pregnant Sprague-Dawley rats for primary fetal mixed neuronal cultures, and PC12s neuronal like cells. Methods BV2 microglia were exposed to ferrous sulfate, lipopolysaccharide, GSK2795039 or GKT137831 and then subsequently co-cultured with neurons or PC12s. Co-culture conditions were maintained for 24 hours and cells were harvested for ROS detection, protein quantification, gene expression, and morphologic alterations by immunocytochemistry. Sample Immortalized and primary rodent cell cultures Analysis Iron dose dependently amplified ROS production among lipopolysaccharide activated microglia. This increased ROS did not modulate gene or protein expression of microglial polarization markers or cytokinechemokine production. It exacerbated neurotoxicity among primary neurons and differentiated PC12s. NOX2 or NOX4 inhibition significantly reversed irons accentuation of ROS production and subsequently ameliorated neurotoxicity. Findings This data substantiates irons contributions to oxidative stress within TBI and provides mechanistic insight into NOX2 and NOX4 contributions to this pathology. This research illustrates the importance of iron mitigation or NOX inhibition as possible future treatment modalities for TBI.
- Medicine and Medical Research