Abstracts

Rationale: The mechanism of status epilepticus (SE)-induced neuronal death in the immature brain is not fully understood. Recently, we reported that neuronal necrosis is a major actor in SE-induced injury in CA1 from P14 rat pups (Niquet et al, 2007). In the present study, we examined the contribution of executioner caspase-3 and initiator caspase-8 and -9 to neuronal injury in CA1 and Dentate Gyrus (DG). Methods: Rat pups were given 3 mEq/kg lithium chloride i.p. on the day before the induction of SE, which was carried out at postnatal day 14 (P14) by subcutaneous injection of 60 mg/kg pilocarpine hydrochloride. Control animals were given an equal volume of saline subcutaneously. Light immunocytochemical studies for active caspase-9, active caspase-3 and caspase-8 were performed by light microscopy 7, 24, 48 and 72 hrs after lithium-pilocarpine SE. Mode of death (apoptotic or necrotic) of active caspase-3-IR neurons was determined by EM postembedding. We determined whether caspase activation contributes to SE-induced hippocampal injury by injecting some animals intraperitoneally with the pan-caspase inhibitor QVD-Oph before SE induction. Neuroprotection was assessed 24 hrs after SE induction by detecting neuronal damage by fluoro-jade B staining. Results: Light immunohistochemical studies showed very little active caspase-3 (C3a), active caspase-9 and caspase-8 in CA1 and DG from control animals. In CA1, 24 h following SE, 19.3 ± 7.9% of dying neurons were cleaved caspase-3-immunoreactive, whereas this number decreased to 10.3 ± 3.7% and 9.6 ± 3.4% 48 and 72 h after SE, respectively. By EM, forty-eight out of 50 immunoreactive CA1 neurons had a necrotic morphology characterized by cytoplasmic and mitochondrial swelling, and pyknosis of the nucleus. In DG, the number of C3a-immunoreactive neurons was significantly increased 7 and 24 hrs. Many C3a-IR DG cells had large round clumps of chromatin (42.5% at 7 h and 45% at 24 h after SE) or displayed chromatin margination (17.9% at 7 h and 39% at 24 h after SE), both features are suggestive of apoptosis. We investigated the contribution of initiator caspases to hippocampal neuronal death following SE. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation, and pretreatment with the pan-caspase inhibitor Q-VD-OPh provided neuroprotection (40% reduction of CA1 injured cells), showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of cleaved caspase-9, but not caspase-8, was detected following SE in apoptotic dentate gyrus neurons. Conclusions: These results suggest that, in cells which are in series as parts of the excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms: necrosis mediated by activation of caspase-8 and -3 in CA1, and apoptosis with caspase-3 and -9 activation in dentate gyrus.