Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice

Abstract

IL-1beta and its endogenous receptor antagonist (IL-1Ra) are rapidly induced by seizures in the rodent hippocampus. Exogenously applied IL-1beta prolongs seizures in an IL-1R type I-mediated manner. This effect depends on N-methyl-d-aspartate receptor activation. We report here that intrahippocampal application of recombinant IL-1Ra or its selective endogenous overexpression in astrocytes under the control of glial acidic fibrillary protein promoter potently inhibits motor and electroencephalographic seizures induced by bicuculline methiodide in mice. Accordingly, transgenic mice show a reduced seizure-related c-fos mRNA expression in various forebrain areas compared with their wild-type littermates. Recombinant IL-1Ra was ineffective in mice deficient in IL-1R type I, having per se a delayed onset to generalized convulsions. These results demonstrate that IL-1Ra mediates potent anticonvulsant effects acting on IL-1R type I and suggest that the balance between brain IL-1beta and IL-1Ra represents a crucial mechanism to control seizure generalization.

Figure 1

High-magnification photomicrographs showing IL-1β (A and B) and IL-1Ra (C and D) immunoreactivity in the hippocampus of representative B6/CBA F₁ mice locally injected with 0.08 nmol bicuculline methiodide (B and D) compared with vehicle-injected controls (A and C). A–D depict corresponding areas of the molecular layer of the dentate gyrus in the injected hippocampus. IL-1β (B) and IL-1Ra (D) staining was enhanced in cells with glial morphology 2 and 4 h after bicuculline injection respectively (arrowheads). No staining was apparent in vehicle-injected mice (A and C) or in mice receiving heat-inactivated cytokines (not shown). A similar immunocytochemical pattern of induction was observed after bicuculline-induced seizures in wild-type SV/129 littermate mice of IL-1R-type I knockout mice (see Table 2) compared with their respective vehicle-injected controls. (Bar = 100 μm.)

Figure 2

Effects of unilateral intrahippocampal injection of 0.3 nmol IL-1Ra (a–c) or 3 pmol IL-1β (d–f) on motor seizure response of B6/CBA F₁ mice to bicuculline methiodide. Data are the mean ± S.E.M. This strain of mice was the same used for engineering transgenic mice overexpressing the human secretable form of IL-1Ra. The anticonvulsant effect of IL-1Ra is denoted by the significant delay in the onset of motor seizures (a) and the reduction in their duration (b and c). All 13 mice in the control group showed both clonic and tonic seizures, whereas 2 mice of 14 did not show tonic seizures after IL-1Ra. The proconvulsant effect of IL-1β is depicted by earlier onset of clonic seizures (d) and by the increase in the duration of motor seizures (e and f). All nine mice in each experimental group showed both clonic and tonic seizures. The proconvulsant effect of IL-1β was assessed by using a submaximal convulsant dose of bicuculline (0.06 nmol) compared with the dose of 0.08 nmol used for testing the anticonvulsant activity of IL-1Ra. Control mice are injected with the corresponding heat-inactivated cytokine before bicuculline methiodide, and they do not differ from B6/CBA F₁ mice (n = 6) receiving vehicle (sterile saline) before bicuculline methiodide. Time in clonus or in tonus was reckoned by adding the duration of each convulsive episode occurring during the 120-min observation period. **, P < 0.01 by Student's t test vs. respective controls.

Figure 3

C-fos mRNA expression after intrahippocampal injection of bicuculline methiodide in B6/CBA wild-type and transgenic mice overexpressing IL-1Ra in astrocytes (GILRA2). First column: C-fos was widely and massively expressed in the central nervous system after injection of bicuculline methiodide in wild-type B6/CBA mice (n = 5) (A). In contrast, mice overexpressing IL-1Ra (GILRA2, n = 7) showed no induction (B, n = 4) or considerably milder induction (C, n = 3) in most forebrain areas, particularly in the neocortex and hippocampus. No specific hybridization signal was found in naive mice (not shown). In A, solid lines in the neocortex and arrows in the hippocampus represent the areas where the hybridization signal was quantified (see text). Second column: Representative EEG tracings of the hippocampus in each row depict epileptic activity after injection of bicuculline methiodide in the corresponding wild-type (A) and GILRA2 mice (B and C) (see Table 1 for quantification of motor seizures). (a) Baseline recording before bicuculline injection; (b) ictal activity recorded in wild-type mice (A) was absent in one GILRA2 (B), whereas it was significantly reduced in number and duration in the remaining animals; (c) interictal spiking was interposed between seizures in wild-type mice (A), and it was present throughout the EEG recording in GILRA2 mice (C). Lower and upper traces in each cluster are the injected and contralateral hippocampus, respectively. (Horizontal bar = 10 sec; vertical bar = 100 μV.)