Elsevier

Neurobiology of Disease

Volume 17, Issue 3, December 2004, Pages 385-402
Neurobiology of Disease

Temporal patterns of the cerebral inflammatory response in the rat lithium–pilocarpine model of temporal lobe epilepsy

https://doi.org/10.1016/j.nbd.2004.07.023Get rights and content

To better understand the role of inflammatory responses in temporal lobe epilepsy, we characterized Interleukin1-β (IL1-β), Nuclear Factor-κB (NF-κB), and Cyclooxygenase-2 (COX-2) expression together with neurodegeneration in the rat lithium–pilocarpine model. The immunohistochemical expression of IL1-β, NF-κB, and COX-2 started by 12 h post-injection, persisted for 24 h (status epilepticus period), and returned to basal levels by 3 and 6 days (latent period). The regional distribution of IL1-β, NF-κB, and COX-2 occurred mainly in structures prone to develop neuronal damage. Using double-staining protocols, we detected IL1-β expression in glial cells, COX-2 expression in neurons, and NF-κB in both cell types. The presence of Fluoro-Jade-B-positive degenerating neurons was associated with IL1-β, NF-κB, and COX-2 proteins expression during status epilepticus but not during the latent period while neurons were still degenerating. These data suggest that seizure-related IL1-β, NF-κB, and COX-2 expression may contribute to the pathophysiology of epilepsy by inducing neuronal death and astrocytic activation.

Introduction

Status epilepticus (SE) induced in the rat by the systemic injection of pilocarpine, a non-subtype-specific partial muscarinic agonist, alone or combined with lithium, shows similarities to human temporal lobe epilepsy (Dubé et al., 2001, Persinger et al., 1988, Turski et al., 1989). Extended neuronal loss and reactive gliosis occur mainly in limbic structures including the hippocampus, amygdala, septum, thalamus, and cerebral cortex (Dubé et al., 2001, Fujikawa et al., 1999, Turski et al., 1989). The acute phase of SE is followed by a latent period characterized by cellular reorganization, before the onset of spontaneous recurrent seizures (Dubé et al., 2001, Roch et al., 2002, Turski et al., 1989).

The identification of signaling molecules expressed during epileptogenesis may help to understand the processes involved in brain damage. Proinflammatory cytokines are key mediators of cellular communication in both physiologic and pathophysiologic states. There is accumulating evidence that among the proinflammatory cytokines, interleukin 1-β (IL1-β) plays a significant role as an effector of the clinical and pathological features of various brain diseases (Rothwell and Hopkins, 1995). Intracerebroventricular administration of recombinant human IL1-β, at the time of experimental traumatic, ischemic or excitotoxic brain injury in the rat, increases the resulting volume of cell death and edema (Patel et al., 2003, Rothwell and Luheshi, 2000, Yamasaki et al., 1995). In contrast, inhibition of the expression or action of endogenous IL1-β by administration of the recombinant interleukin receptor antagonist (IL-1ra) or antibodies against IL1-β markedly reduces the volume of the injury caused by excitotoxicity, trauma, or ischemia (Patel et al., 2003, Yamasaki et al., 1995, Yang et al., 1997). The involvement of IL1-β in the pathology of epilepsy has been repeatedly suggested. IL1-β displays neurotoxic (Jankowsky and Patterson, 2001, Panegyres and Hughes, 1998) and proconvulsant effects in experimental models of SE (Vezzani et al., 1999, Vezzani et al., 2000). Seizures induced by systemic or intra-hippocampal kainate injection, or electrical stimulation enhanced IL1-β, IL1-α protein, and gene expression (De Simoni et al., 2000, Eriksson et al., 1998, Minami et al., 1990, Minami et al., 1991, Nishiyori et al., 1997, Oprica et al., 2003). Seizure activity was worsened by intracerebral injection of the cytokine (Oprica et al., 2003, Vezzani et al., 1999). In addition, polymorphisms in the IL1-β promoter region have been identified at a significantly greater frequency in patients with temporal lobe epilepsy (Kanemoto et al., 2000).

IL1-β has the ability to trigger the transcription of factors like nuclear factor-κB (NF-κB) (Moynagh et al., 1993) that in turn may contribute to the damage induced by SE. NF-κB regulates the expression of many genes mediating the inflammatory responses in CNS diseases (Baeuerle and Baltimore, 1996, Kaltschmidt et al., 1997). NF-κB activity is increased mainly in neurons and glial cells in neurodegenerative disorders (Hunot et al., 1997, Kaltschmidt et al., 1997, Lukiw and Bazan, 1998), models of epilepsy (Lerner-Natoli et al., 2000, Matsuoka et al., 1999, Rong and Baudry, 1996, Unlap and Jope, 1995, Won et al., 1999), and hippocampal tissue from humans with TLE (Crespel et al., 2002). Excitotoxic neuronal death is blocked by pharmacological agents known to inhibit NF-κB activation (Grilli et al., 1996), supporting the hypothesis that NF-κB activation in neurons may be an initiator and regulator of death (Baeuerle and Baltimore, 1996, Grilli et al., 1996). However, cell culture studies suggest that the activation of NF-κB expression may prevent neuronal apoptosis (Mattson et al., 1997, Taglialatela et al., 1997).

The identification of target genes for NF-κB may help to determine their contribution to damage induced by SE. One of the genes that could activate NF-κB in response to epileptic preconditioning is cyclooxygenase-2 (COX-2), the limiting enzyme in prostaglandin synthesis. COX-2 expression is induced in hippocampus by kindling (Tu and Bazan, 2003) and kainate seizures (Hirst et al., 1999, Sandhya et al., 1998) and in the genetically susceptible El mouse (Okada et al., 2001). The COX-2 protein contributes to NMDA-mediated death in cultured neurons (Hewett et al., 2000) and transgenic mice overexpressing neuronal COX-2 are more susceptible to kainate excitotoxicity (Kelley et al., 1999), suggesting that induction of COX-2 could be responsible for tissue damage occurring during seizures. However, the administration of COX-2 inhibitors led to conflicting results in kainate-induced seizures with hippocampal neuroprotection in one study (Kunz and Oliw, 2001), and enhanced damage, and no effect or decreased seizure activity in others studies (Baran et al., 1994, Baik et al., 1999).

To study the potential contribution of IL1-β, NF-κB, and COX-2 during the early genesis of temporal lobe epilepsy, we investigated (1) the timing of appearance of IL1-β, NF-κB, and COX-2 expression after SE induced in the rat by the systemic injection of lithium and pilocarpine; (2) the regional distribution of IL1-β, NF-κB, and COX-2 compared with the pattern of the neuronal degeneration; and (3) the type of cells expressing these factors.

Section snippets

Lithium–pilocarpine-induced status epilepticus

Adult Sprague–Dawley rats (Janvier Breeding Center, Le Genest St-Isle, France) were housed in controlled standard conditions (12 h light/dark cycle, lights on at 7:00 a.m.), with food and water available ad libitum. All animal experimentation was performed in accordance with the rules of the European Community Council Directive of November 24, 1986 (86/609/EEC), and the French Department of Agriculture (License Number 67–97).

Rats were injected with lithium–saline (i.p., 3 meq/kg, Sigma, St.

Time of appearance and regional distribution of IL1-β, NF-κB, and COX-2 and correlation with the pattern of neuronal degeneration

No IL1-β staining was detectable in the brain of lithium–saline rats (Figs. 1A,B) while a few scattered cells weakly positive for NF-κB (Figs. 2A,B) and COX-2 (Figs. 3A,B) were present mainly in cerebral cortex and hippocampus (Table 1, Table 2, Table 3). No detectable Fluoro-Jade B staining was seen at any time point in control animals (Figs. 5A,B).

At 4 h after SE onset, no IL1-β staining was detectable in any brain region (Table 1), while a few scattered cells weakly positive for NF-κB were

Discussion

The present results demonstrate transient IL1-β, NF-κB, and COX-2 expression during the acute phase of SE in the lithium–pilocarpine model of temporal lobe epilepsy. IL1-β expression in glial cells (astrocytes and microglia), COX-2 expression in neurons, and NF-κB expression in both neurons and astrocytes were induced in close temporal and anatomical association with the occurrence of neuronal degeneration. These findings suggest that these inflammation mediators may contribute to

Acknowledgments

We thank Dr. G. Bonvento and Dr. P. Bernard for their contribution to the study.

This work was supported by Fondation de l'Avenir, Grant number ET1-311, the Conseil Régional Champagne-Ardennes and the Institut National de la Santé et de la Recherche Médicale (U 405).

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