Phospholipase A₂-induced neurotoxicity in vitro and in vivo in rats

The present study evaluated the neurotoxic potential of phospholipase A₂ (PLA₂) in vitro (primary neuronal cultures) and in vivo (EEG and behavior) rat models of CNS excitability. In vitro, PLA₂ (0.0038-5.8 nM) or melittin (a potent activator of endogenous PLA₂; 100-5000 nM), were highly neurotoxic, causing approximately 500 units/ml LDH release. The neurotoxic EC₅₀s for PLA₂ and melittin were 1.8 (1.4-2.3) and 848 (501-1280) nM, respectively. Neurotoxic concentrations of PLA₂ stimulated neuronal release of [³H]AA. Preliminary in vitro experiments evaluating changes in neuronal calcium flux indicated that PLA₂ caused transient, and melittin sustained, increases in [Ca²⁺]_i. In vivo, PLA₂ (0.5-5 μg i.c.v.) or melittin (2.5-20 μg i.c.v.) produced nonconvulsive EEG seizures, which generalized to status epilepticus. While the onset of seizure development was markedly delayed for PLA₂ (1.5-4.5 h), the seizure inducing effects of melittin were evident within 3.5 ± 0.2 min and more severe. Both PLA₂ and melittin were lethal, exhibiting LD₅₀s of 0.62 μg and 8.4 μg, respectively. Pretreatment with (+)-MK801 (5 μg, i.c.v.) significantly attenuated melittin, but not PLA₂, in vivo neurotoxicity. PLA₂ induced neuropathology in surviving rats revealed extensive cortical and subcortical injury to forebrain neurons and fibre pathways. Collectively, these results demonstrate the potent neurotoxic potential of PLA₂, the delayed clinical nature of its in vivo neurotoxicity and the applicability of these model systems to future studies on mechanisms of PLA₂ neurotoxicity and the development of potential PLA₂ antagonists.