BACKGROUND AND PURPOSE: Acetylsalicylic acid (ASA) is preventive against stroke and protects against focal brain ischemia in rats. We studied the mechanisms of the manner in which ASA provides neuroprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Spinal cord cultures exposed to 20 hours of hypoxia followed by reoxygenation were treated with a vehicle, ASA or inhibitors of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases p38 MAPK and ERK1/2, or an N-methyl-d-aspartic acid (NMDA) receptor antagonist. Cell viability was assessed by LDH release measurement and cell counts. Prostaglandin production was measured by enzyme immunoassay, MAPK signaling by immunoblotting, and DNA binding of nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) by electrophoretic mobility shift assay. RESULTS: One to 3 mmol/L ASA inhibited H/R-induced neuronal death when present during H/R but not when administered only for the reoxygenation period. Prostaglandin E2 production was very low and was not altered by ASA. The AP-1 and NF-kappaB DNA binding activities increased after H/R. ASA increased the H/R-induced AP-1 binding but had no effect on NF-kappaB binding. H/R induced a sustained ERK1/2 activation followed by neuronal death, whereas no changes in p38 or c-Jun N-terminal kinase were detected. ASA strongly inhibited this ERK1/2 activation. PD98059, an ERK1/2 inhibitor, was also neuroprotective, prevented H/R-induced ERK1/2 activation, and had no effect on NF-kappaB binding activity. Inhibition of NMDA receptors, iNOS, or p38 MAPK did not provide neuroprotection. CONCLUSIONS: Inhibition of the sustained activation of ERK1/2 may partially contribute to neuroprotection achieved by ASA against H/R injury.
BACKGROUND AND PURPOSE:Acetylsalicylic acid (ASA) is preventive against stroke and protects against focal brain ischemia in rats. We studied the mechanisms of the manner in which ASA provides neuroprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Spinal cord cultures exposed to 20 hours of hypoxia followed by reoxygenation were treated with a vehicle, ASA or inhibitors of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases p38MAPK and ERK1/2, or an N-methyl-d-aspartic acid (NMDA) receptor antagonist. Cell viability was assessed by LDH release measurement and cell counts. Prostaglandin production was measured by enzyme immunoassay, MAPK signaling by immunoblotting, and DNA binding of nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) by electrophoretic mobility shift assay. RESULTS: One to 3 mmol/L ASA inhibited H/R-induced neuronal death when present during H/R but not when administered only for the reoxygenation period. Prostaglandin E2 production was very low and was not altered by ASA. The AP-1 and NF-kappaB DNA binding activities increased after H/R. ASA increased the H/R-induced AP-1 binding but had no effect on NF-kappaB binding. H/R induced a sustained ERK1/2 activation followed by neuronal death, whereas no changes in p38 or c-Jun N-terminal kinase were detected. ASA strongly inhibited this ERK1/2 activation. PD98059, an ERK1/2 inhibitor, was also neuroprotective, prevented H/R-induced ERK1/2 activation, and had no effect on NF-kappaB binding activity. Inhibition of NMDA receptors, iNOS, or p38MAPK did not provide neuroprotection. CONCLUSIONS: Inhibition of the sustained activation of ERK1/2 may partially contribute to neuroprotection achieved by ASA against H/R injury.