Using immunocytochemistry and ELISA, we investigated the production of interleukin (IL)-1␤ in the rat hippocampus after focal application of kainic acid inducing electroencephalographic (EEG) seizures and CA3 neuronal cell loss. Next, we studied whether EEG seizures per se induced IL-1␤ and microglia changes in the hippocampus using bicuculline as a nonexcitotoxic convulsant agent. Finally, to address the functional role of this cytokine, we measured the effect of human recombinant (hr)IL-1␤ on seizure activity as one marker of the response to kainate.Three and 24 hr after unilateral intrahippocampal application of 0.19 nmol of kainate, IL-1␤ immunoreactivity was enhanced in glia in the injected and the contralateral hippocampi. At 24 hr, IL-1␤ concentration increased by 16-fold ( p Ͻ 0.01) in the injected hippocampus. Reactive microglia was enhanced with a pattern similar to IL-1␤ immunoreactivity. Intrahippocampal application of 0.77 nmol of bicuculline methiodide, which induces EEG seizures but not cell loss, enhanced IL-1␤ immunoreactivity and microglia, although to a less extent and for a shorter time compared with kainate. One nanogram of (hr)IL-1␤ intrahippocampally injected 10 min before kainate enhanced by 226% the time spent in seizures ( p Ͻ 0.01). This effect was blocked by coinjection of 1 g (hr)IL-1␤ receptor antagonist or 0.1 ng of 3-((ϩ)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate, selective antagonists of IL-1␤ and NMDA receptors, respectively.Thus, convulsant and/or excitotoxic stimuli increase the production of IL-1␤ in microglia-like cells in the hippocampus. In addition, exogenous application of IL-1␤ prolongs kainateinduced hippocampal EEG seizures by enhancing glutamatergic neurotransmission.

IL-1␤ and its endogenous receptor antagonist (IL-1RaIL-1␤ appears to be involved in neuronal network excitability because it affects the turnover and release of various neurotransmitters (1) and the expression of neuropeptides and neurotrophic factors (3-5) and alters synaptic transmission and ionic currents (6-9) in several rodent forebrain regions.Convulsant stimuli increase the production of IL-1␤, its naturally occurring receptor antagonist (IL-1Ra), and IL-1R type I and II predominantly in glia in rodent central nervous system within hours of seizure induction (10-15).We recently showed that IL-1␤ prolongs hippocampal electroencephalographic (EEG) seizures in a N-methyl-D-aspartate receptor-dependent manner, and this action was blocked by .In this study, we investigated whether IL-1Ra has anticonvulsant properties in rodents. We found that intracerebral application of recombinant IL-1Ra or its endogenous overexpression in astrocytes potently inhibited behavioral and EEG seizures induced by bicuculline methiodide in mice. This effect was mediated specifically by IL-1R type I, because IL-1Ra was ineffective in knockout mice deficient in these receptors.Thus, the functional interaction between brain-born IL-1␤ and IL-1Ra during seizures, (i) may play a critical role in the physiopathological functions of IL-1␤, and (ii) may significantly affect the maintenance and spread of seizures. Materials and MethodsAnimals. Procedures involving animals and their care were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (4D. L. N. 116, Gazzetta Ufficiale, supplement 40, 18-2-1992 and European

Limbic status epilepticus was induced in rats by unilateral 60-min electrical stimulation of the CA3 region of the ventral hippocampus. As assessed by RT-PCR followed by Southern blot analysis, transcripts of interleukin-1beta, interleukin-6, interleukin-1 receptor antagonist and inducible nitric oxide synthase were significantly increased 2 h after status epilepticus in the stimulated hippocampus. Induction was maximal at 6 h for interleukin-1beta (445%), interleukin-6 (405%) and tumour necrosis factor-alpha (264%) and at 24 h for interleukin-1 receptor antagonist (494%) and inducible nitric oxide synthase (432%). In rats with spontaneous seizures (60 days after status epilepticus), interleukin-1beta mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1beta, interleukin-6 and tumour necrosis factor-alpha was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1beta immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 microg/3 microL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-alpha content in the hippocampus measured 18 h after status epilepticus. Thus, the induction of spontaneously recurring seizures in rats involves the activation of inflammatory cytokines and related pro- and anti-inflammatory genes in the hippocampus. These changes may play an active role in hyperexcitability of the epileptic tissue.

Summary: Purpose:We investigated the changes in the expression of proinflammatory cytokines and related molecules in the rodent hippocampus after the induction of limbic seizures. We then studied the effects of pharmacologic intervention on the interleukin (IL)-1 system on limbic seizures and the susceptibility to seizures of transgenic mice overexpressing the naturally occurring antagonist of IL-1 (IL-1Ra) in astrocytes.Methods: Limbic seizures were induced in rodents by intrahippocampal injection of kainic acid or bicuculline methiodide or by electrical stimulation of the hippocampus causing status epilepticus (SE). Seizure activity was recorded by EEG analysis and behavioral observation according to Racine's scale. Cytokine expression in the hippocampus was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot quantification of the various messenger RNAs (mRNAs) and by immunocytochemistry.Results: We found that limbic seizures rapidly and transiently enhanced IL-1␤, IL-6, and tumor necrosis factor (TNF)-␣ mRNA in the hippocampus with a peak effect at 6 h after SE. Immunoreactivity of the various cytokines was increased in glia. The increase of IL-1Ra was delayed because the peak effect was observed at 24 h after SE. Moreover, IL-1Ra was not produced in large excess, as during peripheral inflammation but in a molar ratio to IL-1␤ of 1:1. Intrahippocampal injection of IL-1␤ worsened seizure activity, whereas IL-1Ra was a powerful anticonvulsant in various models of limbic seizures. Transgenic mice overexpressing IL-1Ra in astrocytes were less sensitive to bicuculline-induced seizures.Conclusions: This study shows that limbic seizures in rodents rapidly and reversibly induce proinflammatory cytokines in glia and suggests that changes in the IL-1Ra/IL-1␤ ratio in brain may represent an effective physiopathologic mechanism to control seizures.