Short CommunicationEndogenous neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy
Introduction
Cholesterol side-chain cleavage cytochrome P450 (P450scc) is the rate-limiting enzyme for steroidogenesis in all endocrine tissues. This enzyme, which converts cholesterol to pregnenolone, is also present in the central nervous system (CNS) where it plays a key role in the local synthesis of steroids, including GABAA receptor modulatory neurosteroids (Mellon and Griffin, 2002). These steroids potentiate GABA-activated chloride currents, thereby enhancing GABA-mediated inhibition (Lambert et al., 2003). Neurosteroids have a variety of CNS actions related to their effects on the GABAA receptor, including protective activity in diverse experimental seizure models (Rogawski and Reddy, 2004). Therefore, neurosteroids may represent endogenous modulators of seizure susceptibility. Such endogenous GABAA receptor modulatory neurosteroids include allopregnanolone, allotetrahydrodeoxycorticosterone, androstanol and androsterone, which are derived from their parent steroid hormones progesterone, deoxycorticosterone and testosterone by reduction at the 5- and 3-positions on the A-ring. The initial 5α-reduction is rate limiting for neurosteroid synthesis and can be blocked by the azasteroid finasteride, which prevents the anticonvulsant activity of the parent steroids progesterone and deoxycorticosterone (Rogawski and Reddy, 2004). In addition, it has recently been reported that finasteride aggravated seizures in a patient taking progesterone for the treatment of catamenial epilepsy (Herzog and Frye, 2003).
Glial cell reaction is known to accompany brain damage caused by status epilepticus (SE) (Represa et al., 1995, Belluardo et al., 1996). This phenomenon has been mainly characterized in the CA3 subfield of the hippocampus, a region that is vulnerable to damage in several animal models of temporal lobe epilepsy (TLE) (Clifford et al., 1987, Represa et al., 1995). Interestingly, glial cell reaction develops in the first week after SE and slowly resolves in the following weeks (Represa et al., 1995, Belluardo et al., 1996, Borges et al., 2003). Thus, the time course of glial cell reaction overlaps the latent period of epileptogenesis, the interval following SE prior to the onset of spontaneous seizures (Leite et al., 1990). Glia are the most active steroidogenic cells in the brain inasmuch as they contain a greater diversity and express higher levels of steroidogenic enzymes than neurons. In particular, astrocytes and oligodendrocytes and to a lesser extent neurons express P450scc. Here, we sought to determine whether a link existed between glial activation, neurosteroids and seizure development in the latent period. To this end, we studied (i) the time course of P450scc expression in the pilocarpine model of TLE, (ii) the relationship between P450scc and the time of appearance of spontaneous seizures and, finally, (iii) the effects of finasteride on the duration of the latent period.
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Animals and treatments
Male Sprague–Dawley rats (270–300 g, Harlan Italy, S. Pietro al Natisone, Italy) surviving (n = 157) pilocarpine-induced SE were used in four different experiments. Pilocarpine treatment was as described by Biagini et al. (2001). To prevent the effects caused by peripheral muscarinic receptor stimulation, we treated rats with subcutaneous (s.c.) scopolamine methylnitrate (1 mg/kg) 30 min before intraperitoneal (i.p.) pilocarpine (380 mg/kg). Seizure activity was scored according to the scale of
Results
P450scc immunoreactivity was studied in the CA3 strata radiatum and lacunosum-moleculare at intervals following a sustained (4–6 h) episode of SE induced by pilocarpine. As shown in Fig. 1, P450scc immunoreactivity in presumptive glial cells increased and then returned toward control values during the 3-week period following SE. Specifically, there was a doubling in cell counts by day 1 and a sustained tripling from days 3 to 7. Even at 21 days after SE, the number of P450scc-positive glial
Discussion
Our study demonstrates that P450scc, a key enzyme in steroid synthesis, is upregulated in the CA3 hippocampal subfield following pilocarpine-induced SE. These changes occurred mainly in glial cells, although there was also a transient increase in neurons. The induction of P450scc was greater with more prolonged episodes of SE. Similar increases in P450scc staining were evident in other limbic areas, including the dentate gyrus, subiculum and entorhinal cortex, in the pilocarpine-treated rats.
Acknowledgments
This work was supported by the Italian Ministry of Education, University and Research (Fondo per gli Investimenti della Ricerca di Base RBNE01NR34_011, Research Project of Relevant National Interest 2003060538_003), Pierfranco and Luisa Mariani Foundation (R-06-50) and the Canadian Institutes of Health Research (Grant MT-8109).
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