Arachidonic Acid (AA) activates NF-κB and disrupts intracellular calcium homeostasis in cultured spinal cord neurons

Spinal cord trauma can cause a marked release of free fatty acids from cell membranes, which may lead to secondary damage to spinal cord neurons (SCN). A major fatty acid released during trauma is AA. In this study, we investigated the effect of AA on possible compromised functions of SCN. SCN were isolated from spinal cords of fetal mice and maintained in Neurobasal medium enriched with N2 supplement. All experiments were done after 3 weeks following cell seeding. Cells were treated for 2 hours with increasing concentrations of AA (0.01-10 μM). Cellular oxidative stress was measured using DCF fluorescence. Intracellular calcium levels were measured in SCN loaded with fura-2AM. NF-κB in nuclear extracts was determined by electrophoretic mobility shift assay. IκB in cytosol was estimated by Western blotting. Oxidative stress was increased over 20% and 50% in cells exposed to 1 and 10 μM of AA, respectively. In addition, intracellular calcium concentration was significantly elevated in cells treated with AA. NF-κB was activated and levels of IκB were decreased in a dose-dependent manner in SCN exposed to AA. These results strongly support our hypothesis that free fatty acids may significantly contribute to the tissue injury observed following spinal cord trauma.