Abstracts

RATIONALE: Our previous evidence has shown that limbic seizures in adult rats induce a rapid and reversible cytokines synthesis in the hippocampus both in microglia and astrocytes. This occurred at a larger extent when seizures were associated to neuronal loss. Intracerebral application of IL-1beta exacerbates seizures while TNF-alpha has anticonvulsant activity.
The aim of this study was to assess whether cytokines synthesis and glia activation are triggered by seizures in immature brain. We also compared the pattern of cytokine induction with the occurrence of age-dependent seizure-induced neuronal cell injury using 10 and 15 day-old and pre-adolescent 21 day-old rats.
METHODS: Limbic seizures were induced in 10 and 15 day-old rats by systemic injection of 1.3 and 5 mg/kg kainic acid respectively while 10 mg/kg kainate was used in 21 day-old rats. Behavioral seizures resembling generalized clonic convulsions lasted for 90 min on average in the different age groups and had an onset time of about 15 min in the 10 day-old group and of 20-30 min in the other groups.
For RT-PCR measurements of cytokines mRNA (IL-1beta, IL-6, TNF-alpha, IL-1Ra), rats were killed by decapitation 2-24 h after the end of seizures and their hippocampi were frozen at [ndash]70[degree]C until assay. Different groups of rats were killed by transcardial perfusion using buffered saline followed by 4% paraformaldehyde and adjacent 40 [mu]m coronal forebrain sections were cut at a cryostat for histochemical detection of neuronal injury by Fluoro Jade and specific astrocyte (GFAP) and microglia (OX-42) markers.
RESULTS: In 10 day-old pups, seizures induced a decrease in IL-1beta, IL-1 receptor antagonist (Ra) and TNF-alpha mRNAs within 24 h from seizure onset while IL-6 mRNA did not change. In 15 day-old rats, IL-1beta did not change at 2 h while a 2.5-fold increase was found 4 h after seizures then returning to basal level by 24 h. None of the other cytokines was modified in these rats. In 21 day-old rats, the pattern of cytokine changes was similar to the adult (3 month-old) rats. Thus, IL-1beta and IL-1Ra were increased by 4- and 2-fold respectively, TNF-alpha by 1.5-fold and IL-6 by 5-fold.
Microglia and astrocytes activation occurred after seizures at all age groups, 2-24 h after seizures particularly in the hippocampus and entorhinal cortex. Astrocytes were mostly activated in CA3 and dentate gyrus at 2 h then in all hippocampal subfields at later times.
Fluoro Jade staining revealed a few positive neurons in the hilus of 15 day-old rats, 24 h after seizures. In 21 day-old rats, various positive neurons were localized in the CA3 pyramidal layer, hilus and superficial layers of the temporal cortex.
CONCLUSIONS: Our evidence indicates that limbic seizures do not induce cytokines in brain until the onset of seizure-dependent neuronal cell injury (i.e. 15 and 21 day-old rats). The lack of cytokines production is not due to functional impairment of glia since both microglia and astrocytes are activated at all ages similarly to adult rat brain.
We suggest that the lack of proinflammatory cytokines induction in the immature brain during seizures may play a role in its refractoriness to nerve cell damage.
[Supported by: CURE, Telethon Onlus GPO285/01]