Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death

The present study sought to evaluate the neuroprotective effects of apocynin, an NADPH oxidase assembly inhibitor, on seizure-induced neuronal death. Apocynin, also known as acetovanillone, is a natural organic compound isolated from the root of Canadian hemp (Apocynum cannabium). It has been extensively studied to determine its disease-fighting capabilities and application in several brain insults, such as traumatic brain injury and stroke. Here we tested the hypothesis that post-treatment of apocynin may prevent seizure-induced neuronal death by suppression of NADPH oxidase-mediated superoxide production. Temporal lobe epilepsy (TLE) was induced by intraperitoneal injection of pilocarpine (25 mg/kg) in male rats. Apocynin (30 mg/kg, i.p.) was injected into the intraperitoneal space two hours after seizure onset. A second injection was performed 24 h after seizure. To test whether apocynin inhibits NADPH oxidase activation-induced reactive oxygen species (ROS) production, dihydroethidium (dHEt, 5 mg/kg, i.p.) was injected before onset of seizure and ROS production was detected five hours after seizure onset. Neuronal oxidative injury (4HNE), neuronal death (Fluoro Jade-B), blood brain barrier (BBB) disruption (IgG leak), neurotrophil infiltration (MPO) and microglia activation (CD11b) in the hippocampus was evaluated at three days after status epilepticus (SE). Pilocarpine-induced seizure increased p47 immunofluorescence in the plasma membrane of hippocampal neurons at 12 h post-insult and apocynin treatment prevented this increase. The present study found that apocynin post-treatment decreased ROS production and lipid peroxidation after seizure and decreased the number of degenerating hippocampal neurons. Apocynin also reduced seizure-induced BBB disruption, neurotrophil infiltration and microglial activation. Taken together, the present results suggest that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce seizure-induced neuronal dysfunction.

► NADPH oxidase activation is an important neuron death mechanism after seizure. ► Apocynin reduced seizure-induced ROS production and BBB disruption. ► Apocynin reduced seizure-induced microglial activation and neuron death. ► Post-treatment of apocynin is effective up to two hours after status epilepticus.