Expression and activation of caspase 3 following status epilepticusintherat Susanna Narkilahti, 1 Terhi J. Pirttila È, 1 Katarzyna Lukasiuk, 1 Jarkko Tuunanen, 1 and Asla Pitka Ènen 1,2 1 A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, PO Box 1627, FIN-70 211 Kuopio, Finland 2 Department of Neurology, Kuopio University Hospital, PO Box 1777, FIN-70211 Kuopio, Finland Keywords: amygdala, epileptogenesis, hippocampus, programmed cell death, seizure Abstract It is in dispute whether caspase3 contributes to status epilepticus (SE)-induced cell loss. We hypothesized that caspase3-mediated cell death continues beyond the acute phase of SE. We induced SE with either kainic acid or electrical stimulation of the amygdala in Wistar and Sprague-Dawley rats. Caspase3 immunohistochemistry, Western blot analysis and enzyme activity measurements were used to determine cellular localization and the time course of caspase3 expression and activation. Immunohistochemistry indicated thatcaspase3proteinexpressionincreasedfollowingSE,peakingat16±24h.Cleavageofprocaspase3toactivefragments(p20±17) wasdetected2±7daysafterSE.Caspase3enzymeactivitywaselevatedat8handfurtherincreasedupto19.4-foldat7daysfollowing SE. Activation of caspase3 after SE occurred in the hippocampus and the extrahippocampal temporal lobe but not in the thalamus. Caspase3-immunoreactivecellsrepresentedonlyaminorityofdegeneratingcellsasassessedbyFluoro-JadeBandTUNELstaining. Analysisofdouble-labelledsectionsindicatedthatactivecaspase3waslocatedinastrocytesratherthanneuronsormicroglia.There was increased caspase3 expression in both rat strains, and it was independent of the method used to induce SE. These data demonstrate that caspase3 contributes to the cell death occurring within the ®rst week after SE, but in only a small proportion of degenerating cells. These results suggest that, contrary to expectations, caspase3 inhibitors would have only limited bene®ts in the treatment of SE. Introduction Epilepsies comprise the second most common group of neurologic disorders after stroke. Approximately two-thirds of patients have partial onset of seizures (Houser, 1997) and, in most subjects, epilepsy develops as a consequence of brain damaging insult such as head trauma, stroke or status epilepticus (SE) (Mathern et al., 1995). Neurobiologic changes occurring during epileptogenesis include neu- ronal death, gliosis and axonal and dendritic plasticity, with neuronal death being the most prominent feature in the early phase (for review see Jutila et al., 2002). Therefore, the use of neuroprotectants in the treatment of patients with epileptogenic brain insults is of major clinical interest. For rational neuroprotective drug design, identi®ca- tion of the cell death mechanisms and an understanding of their contribution to overall damage is critical. Recent histologic and magnetic resonance imaging studies indicate that SE-induced damage can continue for weeks (Pitka Ènen et al., 2002). Most of the acute (< 3days) neuronal death has necrotic features (Olney et al., 1974; Fujikawa et al., 2000) but recent data suggest that various caspases and programmed cell death can also contribute to SE-induced damage (Gillardon etal.,1997;Faherty etal., 1999; Ferrer et al., 2000). The major executioner caspase involved in apoptosis is caspase 3 (Nicholson et al., 1995; Ni et al., 1997). The increased expression and activation of caspase3 has been found acutely (< 3days) after SE in rats (Ferrer et al., 2000; Henshall et al., 2000a). Also, patients operated on for drug-refractory epilepsy had elevated levels of procaspase3 and cleaved caspase3 in the temporal cortex (Henshall et al., 2000b). The application of caspase 3 inhibitors reduced SE-induced neuronal loss (Henshall et al., 2000a; Narkilahti et al., 2003) and DNA laddering in the rat hippocampus (Kondratyev & Gale, 2000). Thus, the idea of using caspase3 inhi- bitors as neuroprotectants for antiepileptogenic treatment is attractive and has raised a lot of interest. The proportion of caspase 3-mediated cell death in SE-induced damage, and the time course of its activation beyond the acute phase, however, are unknown. We hypothesized that the contribution of caspase3-mediated cell death to SE-induced damage continues beyond the acute phase of SE. Totestthishypothesisthefollowingquestionswereaddressed:(i)what is the temporal and spatial distribution of caspase 3 expression after SE; (ii) is caspase 3 processed to an active enzyme in vivo; (iii) what is the proportion of caspase 3-mediated cell death in overall SE-induced damage; (iv) in which cell types is active caspase3 expressed; and (v) what is the duration of SE required to induce caspase 3 expression? Because of the possible contribution of genetic background on kainate- induced seizures (Schauwecker, 2002), experiments were performed in two rat strains. Further, to avoid the bias related to the use of just one model, SE was induced either chemically or electrically. Materials and methods Animals Adult male Harlan Sprague-Dawley rats (Netherlands) or male Han- Wistar rats (Kuopio University) weighing 300±350g were housed in individual cages at a temperature of 19±21 8C, humidity 50±60% and European Journal of Neuroscience, Vol. 18, pp. 1486±1496, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02874.x Correspondence: Dr Asla Pitka Ènen, 1 A.I. Virtanen Institute for Molecular Sciences, as above. E-mail: asla.pitkanen@uku.fi Received 11 June 2003, accepted 4 July 2003