Hippocampal Programmed Cell Death after Status Epilepticus: Evidence for NMDA-Receptor and Ceramide-Mediated Mechanisms *†§Mohamad A. Mikati, *†§Ralph J. Abi-Habib, ‡Marwan E. El Sabban, *†Ghassan S. Dbaibo, *§Rana M. Kurdi, *§Mohamad Kobeissi, *§Firas Farhat, and †Wissal Asaad Departments of *Pediatrics, †Biochemistry, ‡Human Morphology, and §Adult and Pediatric Epilepsy Program, Faculty of Medicine, American University of Beirut, Beirut, Lebanon Summary: Purpose: Status epilepticus (SE) can result in acute neuronal injury with subsequent long-term age-dependent behavioral and histologic sequelae. To investigate potential mechanisms that may underlie SE-related neuronal injury, we studied the occurrence of programmed cell death (PCD) in the hippocampus in the kainic acid (KA) model. Methods: In adult rats, KA-induced SE resulted in DNA fragmentation documented at 30 h after KA injection. Cer- amide, a known mediator of PCD in multiple neural and non- neural tissues, increased at 2–3 h after KA intraperitoneal injection, and then decreased to control levels before increasing again from 12 to 30 h after injection. MK801 pretreatment prevented KA-induced increases in ceramide levels and DNA fragmentation, whether there was reduction in seizure severity or not (achieved with 5 mg/kg and 1 mg/kg of MK801, respec- tively). Results: Both ceramide increases and DNA fragmentation were observed after KA-induced SE in adult and in P35 rats. Ceramide did not increase after KA-induced SE in P7 pups, which also did not manifest any DNA fragmentation. Intrahip- pocampal injection of the active ceramide analogue C2- ceramide produced widespread DNA fragmentation, whereas the inactive ceramide analogue C2-dihydroceramide did not. Conclusions: Our data support the hypotheses that (a) N- methyl-D-aspartate–receptor activation results in ceramide in- creases and in DNA fragmentation; (b) ceramide is a mediator of PCD after SE; and (c) there are age-related differences in PCD and in the ceramide response after SE. Differences in the ceramide response could, potentially, be responsible for ob- served age-related differences in the response to SE. Key Words: Kainic acid—Status epilepticus—Programmed cell death—Ceramide—NMDA receptor. Apoptosis is a morphologically distinct type of cell death characterized by cellular shrinkage and condensa- tion; large, round, or crescent-shaped chromatin clumps within the nucleus; and relative preservation of cytoplas- mic organelles (1). Programmed cell death (PCD) refers to an ordered process that leads to cell death through predetermined and reproducible mechanisms that usually require de novo protein synthesis. PCD is characterized by certain biochemical features such as DNA fragmen- tation into oligonucleosomal fragments (2). Although generally used to refer to the same final outcome, apo- ptosis and PCD are not always identical, especially be- cause PCD has recently been shown to occur in neurons with the morphologic features of necrosis (3,4). A num- ber of studies have reported the occurrence of PCD after status epilepticus (SE) in adult rats, in the kainic acid (KA), pilocarpine (PC), and perforant-path stimulation models (5–9). The mechanisms leading to PCD after SE remain largely unknown. Some studies suggested that N-methyl-D-aspartate (NMDA)-receptor activation is necessary for PCD after SE (4,6,10). Others failed to show such an effect (5). The reason for this discrepancy is probably that different studies used different doses of MK801, resulting in different degrees of blockade of NMDA receptors. In addition, none of these studies con- trolled for the severity of seizure activity after MK801 administration, and therefore did not exclude the possi- bility that the observed protective effects of MK801 may be due to decreases in seizure severity per se, and not to NMDA-receptor blockade. The sphingolipid ceramide is a mediator of intracellu- lar signaling pathways of PCD in several cell lines and tissues including neuronal tissues (11–19). The potential role of ceramide in SE-induced PCD has not been pre- viously investigated. Accepted October 27, 2002. Address correspondence and reprint requests to Dr. M. Mikati at Adult and Pediatric Epilepsy Program, Department of Pediatrics, American University of Beirut, 850 Third Avenue, 18th Floor, New York, NY 10022, U.S.A. E-mail: mamikati@aub.edu.lb Epilepsia, 44(3):282–291, 2003 Blackwell Publishing, Inc. © 2003 International League Against Epilepsy 282