Cytosolic phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice

Background: The enzyme cytosolic phospholipase A(2) alpha (cPLA(2)alpha) has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA2a enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA(2)alpha in early ischemic cerebral injury. Methods: Middle cerebral artery occlusion (MCAO) was performed on cPLA(2)alpha(+/+) and cPLA(2)alpha(-/-) mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA(2)alpha, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE(2) content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion. Results: Neuronal cPLA(2)alpha protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE2 concentration were greater in cPLA2a+/+ compared to cPLA(2)alpha(-/-) ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA(2)alpha(+/+) than in cPLA(2)alpha(-/-) brains (+/+: 2.2 +/- 0.3 fold vs. -/-: 1.7 +/- 0.4 fold increase; P < 0.01). The increase in reactive oxygen species following 2 hours of ischemia was also significantly greater in the cPLA(2)alpha(+/+) ischemic core than in cPLA2a-/-(+/+: 7.12 +/- 1.2 fold vs. -/-: 3.1 +/- 1.4 fold; P < 0.01). After 6 hours of reperfusion ischemic cortex of cPLA(2)alpha(+/+), but not cPLA(2)alpha(-/-), had disruption of neuron morphology and decreased PGE2 content. Phosphorylation of the MAPKs-p38, ERK 1/2, and MEK 1/2-was significantly greater in cPLA(2)alpha(+/+) than in cPLA(2)alpha-/ischemic cortex 6 hours after reperfusion. Conclusions: These results indicate that cPLA(2)alpha modulates the earliest molecular and injury responses after cerebral ischemia and have implications for the potential clinical use of cPLA(2)alpha inhibitors.