Several studies report microglial accumulation and activation in the CA1 area in response to transient forebrain ischemia (TFI). Here we examine the possibility that free radicals and chemokines mediate the transient activation of microglia. Free radicals are produced primarily in CA1 pyramidal neurons within 2 h of TFI. Administration of trolox, a vitamin E analog, led to the inhibition of free radical production and recruitment of microglia in the CA1 area. In addition, intrahippocampal injection of Fe 2ϩ triggered free radical production in CA1 neurons, followed by the recruitment and activation of microglial cells into this area. TFI-induced expression of macrophage inflammatory protein-1␣ (MIP-1␣) was increased in CA1 neurons before microglial recruitment, and blocked by trolox. Moreover, the MIP-1␣ level was upregulated in cultured hippocampal neurons exposed to Fe 2ϩ , suggesting an essential role of free radicals in TFIinduced expression of MIP-1␣. Intracerebroventricular injection of vMIP-2 (viral macrophage inflammatory protein-2), a broadspectrum peptide antagonist of chemokine receptors, attenuated microglial recruitment and delayed CA1 neuronal degeneration after TFI. Our data suggest that free radicals produced in CA1 neurons contribute to the recruitment and activation of microglia and neurodegeneration through MIP-1␣ expression.

Excess activation of ionotropic glutamate receptors, primarily N-methyl-D-aspartate (NMDA) receptors and free radicals, evoke nerve cell death following hypoxic-ischemic brain injury in various animal models. However, clinical trials in stroke patients using NMDA receptor antagonists have failed to show efficacy primarily due to the limited therapeutic time window for neuroprotection and a narrow therapeutic index. In comparison, antioxidants prolonged the time window for neuroprotection in animal models of ischemic stroke and showed greater therapeutic potential in clinical trials for ischemic stroke. Excess activation of NMDA receptors and free radicals mediate the two separate pathways of nerve cell death in stroke and a safe and multifunctional drug that can block both routes in the brain will likely provide a better therapeutic outcome in patients with stroke. Derivatives of the lead structures of sulfasalazine and aspirin have led to the discovery of a new molecule, Neu2000, that has demonstrated excellent neuroprotection against NMDA- and free radical-induced cell death. Neu2000 is an NR2B-selective, moderate NMDA receptor antagonist with potent cell-permeable, spin trapping antioxidant action even at nanomolar concentrations. Nonclinical and human phase I studies demonstrated that Neu2000 can be translated to treat patients with stroke with better efficacy and therapeutic time window.