Proinflammatory cytokines and apoptosis following glutamate-induced excitotoxicity mediated by p38 MAPK in the hippocampus of neonatal rats

https://doi.org/10.1016/j.jneuroim.2005.04.025Get rights and content

Abstract

The proinflammatory cytokines TNF-α, IL-1β, and IL-6 rise during neuronal damage and activate the apoptotic mitogen-activated protein kinase p38. We studied apoptosis, the levels of TNF-α, IL-1β, and IL-6, and the cell type producing TNF-α in rats at 8, 10, and 14 days of age after neonatal exposure to glutamate, which induces neuronal damage. TNF-α production was significantly increased by glutamate, but inhibited by SB203580 (a p38 inhibitor). TNF-α, IL-1β, and IL-6 mRNA levels increased, but SB203580 did not modify their expression. Thus, the p38 signaling pathway influences the expression of inflammatory genes and its inhibition may offer anti-inflammatory therapy.

Introduction

Cytokines are involved not only in the immune response, but also in a variety of physiological and pathological processes, including events in the peripheral and the central nervous system (CNS). Thus, they are both immunoregulators and neuromodulators (Szelényi, 2001). Cytokines produced within the CNS might also influence cognition, learning, and memory. This involvement of inflammatory cytokines in neural functions suggests that these cytokines might have an important role in integrating physiological autonomic responses (Szelényi, 2001).

Altered expression of various cytokines in the brain is observed in a variety of CNS disorders, for example Alzheimer's disease (Zhang et al., 2004), multiple sclerosis (Ozenci et al., 2002), viral or bacterial infections (Beadling and Slifka, 2004), ischemia (Yu and Lau, 2000), and various forms of encephalopathies (Campbell et al., 1998). There is evidence that transmitters released from axon terminals without synaptic contact play an important role in communication not only between neurons, but also between nerve terminals and immune cells (Vizi, 2000). Because immune cells are equipped with neurotransmitter and neuropeptide receptors, mediators such as noradrenaline and other monoamines, nucleosides, acetylcholine, and endorphins are able to modulate the cytokine response to a given stimulus (Vizi, 2000).

On the other hand, glutamate toxicity is attributed to excessive stimulation of their receptors mainly NMDA and AMPA subtypes (Arundine and Tymianski, 2003). Several molecules are activated by glutamate via calcium influx, including CaM kinase II, protein kinase C, nitric oxide synthase, and mitogen-activated protein kinase suggesting that p38 might be involved in glutamate neurotoxicity in cerebellar granule cells (Grilli et al., 1996). In addition, it had been demonstrated that p38 was active, and an increased in number of astrocytes following transient and permanent middle cerebral artery occlusion was also shown in the CNS of the rat (Irving et al., 2000). Recent studies from our work group shown an important reactivity of astrocytes and microglia cells in cerebral cortex of adult rats, after neonatal exposure to glutamate as monosodium salt (MSG) to new born rats (Martinez-Contreras et al., 2002). Likewise, an important increase in neuronal death via apoptosis was also shown from postnatal age 8 to 14 it was accompanied with high TNF-α expression and levels under the same model of study (Chaparro-Huerta et al., 2002). This increase in neuronal death was clearly associated with p38 via activation through high ATF-2 levels, mediating changes in the AMPA receptor composition with lower GluR2 expression in its structure in the cerebral cortex (Rivera-Cervantes et al., 2004). Thus, there is a relationship between p38 activation and apoptosis, although the activating agents vary (Wang et al., 2003, Takeda and Ichijo, 2002). It is widely known that hippocampus is a region highly sensitive to degenerate under ischemia–hypoxia and glutamate receptors over-stimulation; however, if the some pro-inflammatory cytokines are associated with neuronal death through via p38 activation under an early exposure to high glutamate concentrations at hippocampus of neonatal rats, it is unknown. Therefore, the aim of this work was evaluate the TNF-α, IL-1β and IL-6 expression levels, as well as the cellular localization of TNF-α associated with apoptosis and p38 pathway activation at the hippocampus rats of 8, 10 and 12 days of age under normal conditions and with neuronal damage induced by neonatal exposure to MSG.

Section snippets

Animal experiments

Pregnant Wistar rats were kept under optimal environmental conditions with free access to water and food, under 12–12 h light–dark cycles, at temperatures ranging between 23 °C and 25 °C, and in separate cages. On the day of birth, all litters were adjusted to eight offspring per female. Offspring were given monosodium glutamate (MSG: 4 mg/g body weight) subcutaneously (s.c.) on postnatal days (PD) 1, 3, 5, and 7, according to Beas-Zárate et al. (2001). A group of untreated animals was used as

TNF-α concentrations and mRNA levels

A time–response curve for TNF-α levels was constructed to evaluate the effect of neurotoxicity in the hippocampus induced by neonatal exposure to MSG. TNF-α levels were measured each 6 h after the first dose of MSG was administered, up to PD 8. There was a significant increase in TNF-α concentration at 12 h after the second dose of MSG in comparison with controls (Fig. 1A). These high TNF-α levels remained elevated 60 h after last dose of MSG (Fig. 1A). However, 72 h after TNF-α levels was

Discussion

Glutamate and proinflammatory cytokines have been shown to mediate structural and functional deterioration of the brain, reflected by edema, histological damage, and neurological impairment (Knoblach et al., 1999), and these effects can be influenced pharmacologically (Obrenovitch and Urenjak, 1997). Traumatic brain injury is associated with sustained production and release of the proinflammatory cytokines leading an increase in the extracellular and cerebrospinal fluid (CSF) concentrations (

Acknowledgements

This work was partially supported by CONACyT grant 30901-M to C. B. Z. and scholarship No. 158761 to V. Ch. H.

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