Neuroprotective effect of a mixture of conjugated linoleic acid isomers on neuronal cell death

Glutamate excitotoxicity, which is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors, directly contributes to the neuronal cell loss associated with both acute insults and chronic neurodegenerative disorders. Conjugated linoleic acid (CLA) is a group of dienoic derivatives of linoleic acid shown to have anticarcinogenic and antioxidative activities. To evaluate the effect of a mixture of CLA isomers (cis-9, trans-11 and cis-10, trans-12 octadecadienoic acids) on glutamate- and NMDA-induced excitotoxicity, primary cultures of rat cortical neurons were treated for 15 min with 300 muM glutamate or NMDA in the presence of various concentrations of CLA. After the exposure, cell cultures were maintained at 37°C for 18 h in minimum essential medium supplemented with glucose. Neuronal injury was measured by a colorimetric cell proliferation assay, and a qualitative assessment was made by phase-contrast microscopy. CLA inhibited glutamate- and NMDA-induced neuronal cell death in a concentration-dependent fashion with the most effective dose for neuroprotection being 500 muM These results demonstrate that a mixture of CLA isomers exhibits protective action against glutamate- and NMDA-induced excitotoxicity. To determine the effect of NMDA and non-NMDA receptor antagonists on CLA-induced neuroprotection, rat cortical neurons were incubated with either vehicle or a mixture of CLA isomers in the presence or absence of 2-amino-5-phosphovalerate (AP-5, an NMDA antagonist) or 6-cyano-7nitroquinoxaline-2,3-dione (CNQX, a non-NMDA antagonist) prior to glutamate exposure. The combination of a mixture of CLA isomers with CNQX, but not with AP-5, during the pretreatment prevented cortical neuronal cell death following exposure to glutamate. This result demonstrated that a mixture of CLA isomers might protect neurons from glutamate-induced toxicity by blocking NMDA receptors. The expression of apoptotic genes (bcl-2, bax, bcl-xL, and caspase-3) was analyzed by reverse transcriptase-polymerase chain reaction. A mixture of CLA isomers reduced caspase-3 expression in rat cortical neuronal cells exposed to 300 muM glutamate. CLA exhibits protective action against glutamate-induced toxicity, and the protective action is due to blocking NMDA receptors and to reducing caspase-3 gene expression.