Elsevier

Neurobiology of Disease

Volume 14, Issue 3, December 2003, Pages 494-503
Neurobiology of Disease

Regular article
Glia activation and cytokine increase in rat hippocampus by kainic acid-induced status epilepticus during postnatal development

https://doi.org/10.1016/j.nbd.2003.08.001Get rights and content

Abstract

In adult rats, status epilepticus (SE) induces cytokine production by glia especially when seizures are associated with neuronal injury. This suggests that cytokines may play a role in seizure-induced neuronal damage. As SE-induced injury is age-specific, we used rats of different ages (with distinct susceptibilities to seizure-induced neuronal injury) to elucidate the role of cytokines in this process. Thus, we investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal injury 4 and 24 h following kainic acid-induced SE in postnatal day (PN) 9, 15, and 21 rats. At PN9, there was little activation of microglia and astrocytes at any time point studied. Interleukin-1β (IL), tumor necrosis factor-α (TNF), and IL-6 or the naturally occurring IL-1 receptor antagonist (Ra) mRNA expression did not increase. No evidence of cell injury has been detected. At PN15, immunostaining of microglia and astrocytes was enhanced, but only IL-1β mRNA expression was increased. These changes were observed 4 h after SE. Scattered injured neurons in CA3 and subiculum, but not in any other region, were present 24 h following SE. At PN21, immunostaining of microglia and astrocytes and the mRNA expression of all cytokines studied was significantly increased already 4 h after SE. At 24 h, many injured neurons were present in CA1 and CA3 regions and in 40% of rats in other forebrain areas. These data show that (i) the pattern of glia activation and cytokine gene transcription induced by SE is age-dependent and (ii) neuronal injury in the hippocampus occurs only when cytokines are induced and their synthesis precedes the appearance of neuronal damage. Thus, cytokine expression in immature brain is associated specifically with cell injury rather than with seizures per se, suggesting that proinflammatory cytokines may contribute to the occurence of SE-induced hippocampal damage.

Introduction

Molecules and pathways associated with immune or inflammatory responses have been identified in CNS and are activated in various pathophysiological conditions (Allan and Rothwell, 2001). Among these molecules, cytokines are polypetides mediating interactions among components of the immune system, glia, and neurons.

Proinflammatory and anti-inflammatory cytokines are synthesized by microglia and astrocytes in adult rodent brain within 30 min from seizure onset Minami et al., 1991, Vezzani et al., 1999, Vezzani et al., 1999, De Simoni et al., 2000. The enhanced immunostaining of these molecules in glia is due to neosynthesis as shown by increased levels of their respective transcripts in epileptic tissue Minami et al., 1990, Minami et al., 1990, Yabuuchi et al., 1993, Eriksson et al., 1998, De Simoni et al., 2000, Plata-Salaman et al., 2000. Cytokines are predominantly increased by seizures in the hippocampus where they influence the ongoing epileptic activity Minami et al., 1990, Minami et al., 1990, Eriksson et al., 1998, De Simoni et al., 2000, Vezzani et al., 1999, Vezzani et al., 1999, Vezzani et al., 2000. Their expression is greater in intensity and distribution (i.e., involving entorhinal and temporal cortices) when seizures are associated with neuronal damage, suggesting that neuronal injury and cytokine overexpression are interconnected. In accordance, a role for proinflammatory molecules, and in particular of interleukin-1β (IL), as mediators and modulators of diverse forms of neurodegeneration (i.e., excitotoxic, ischemic, and traumatic) has been well established in mature rat brain Yamasaki et al., 1995, Schobitz et al., 1994, Loddick et al., 1997, Shohami et al., 1999, Barone et al., 1997, Allan and Rothwell, 2001.

In humans and in experimental models of epilepsy, seizure susceptibility and associated neuronal damage are age-dependent. Thus, in the first two postnatal weeks, the rat brain is highly prone to seizure activity but it is relatively resistant to irreversible seizure-induced damage as compared to adult brain Albala et al., 1984, Moshé et al., 1981, Cherubini et al., 1983, Cavalheiro et al., 1987, Moshé et al., 1983, Sperber et al., 1991, Stafstrom et al., 1992, Haas et al., 2001). In immature rats, status epilepticus (SE), kindling or hyperthermia-induced seizures lead to profound yet primarily transient alterations in neuronal structures although long-term effects on neuronal excitability can occur Moshé and Albala, 1982, McCaughran and Schechter, 1982, Okada et al., 1984, Toth et al., 1998, Holmes et al., 1998, Dubé et al., 2000, Meilleur et al., 2000, Lynch et al., 2000, Villeneuve et al., 2000.

The key determinants of the age-depedent occurrence of seizure-induced injury are still unclear. Existing evidence suggests that this is a multifactorial phenomenon. In particular, the presence of immature mossy fibers in hilar region until PN15 (Ribak and Navetta, 1994) and the sustained expression of GluR2 and GABA receptor subunits following kainic acid-induced seizures (Friedman et al., 1997) in the first 2 postnatal weeks have been suggested as having a significant role in the resistance of developing hippocampal neurons to damage following kainic acid-induced SE.

Our hypothesis is that activation of glia and the subsequent production of inflammatory molecules are involved in seizure-induced neuronal damage. To test this, we used rats of different ages [postnatal days (PN) 9, 15, and 21] known to have distinct susceptibility to seizures and neuronal injury. We studied (i) whether microglia and astrocytes are activated after SE induced by kainic acid, because these cells represent the major source of cytokine synthesis in mature rodent brain (Allan and Rothwell, 2001); (ii) whether proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-1Ra) cytokines are induced by SE in immature rat brain; and (iii) if there is a relationship between SE-induced cytokine changes in immature brain and the occurrence of neuronal cell injury.

Our data indicate that sustained seizures activate glia and induce cytokine synthesis in age-dependent manner and suggest that proinflammatory cytokines, in particular IL-1β may play a role in SE-associated neuronal injury during postnatal development.

Section snippets

Experimental animals

Male Sprague–Dawley rats (Charles River, Calco, Italy) of postnatal day (PN) 9, 15, and 21 (with PN 0 defined as the day of birth) were used. All animals were used before weaning. Pups were housed with their dams at constant temperature (23°C) and relative humidity (60%) with a fixed 12 h light–dark cycle and free access to food and water. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L.n.116,

Glia activation by SE during postnatal development

SE increased OX-42 and GFAP immunostaining mainly in the hippocampus with no differences between individual hippocampal regions examined (CA1, CA3, dentate gyrus). SE-induced changes in OX-42 and GFAP immunostaining were age-dependent.

Figure 1 shows OX-42 immunostaining in area CA3 of hippocampi from representative PN9, PN15, and PN21 rats, 4 h and 24 h after SE onset, and their respective controls (n = 5–10 rats each group). Faint immunoreactivity was found in control sections at all

Discussion

Recent findings have shown that IL-1β is produced both by microglia and astrocytes in adult rodent brain in various neuropathological conditions, including seizures. This cytokine, together with other proinflammatory molecules, appears to contribute to the mechanisms mediating excitotoxic and/or apoptotic neuronal cell death (see for review Allan and Rothwell, 2001). Accordingly, IL-1β blockade by intracerebral application of IL-1Ra significantly protects from ischemic, traumatic, and

Acknowledgements

We thank Ms Daniela Moneta for her contribution to this study. This work was supported by CURE, Fondazione Mariani Onlus, the Heffer Family Foundation (AV), and Grants NS-36238 (JV) and NS-20253 (SLM) from the NINDS. SLM is the recipient of the Martin A. and Emily L. Fisher fellowship in Neurology and Pediatrics. MA is the recipient of the F.B. 2001 fellowship.

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