Cyclooxygenase-2 mediates the sensitizing effects of systemic IL-1-beta on excitotoxic brain lesions in newborn mice

Abstract

Epidemiological and experimental data implicate maternal–fetal infection and an associated increase in circulating cytokines in the etiology of cerebral palsy. We have previously shown that pretreatment of newborn mice with systemic interleukin-1-beta exacerbates ibotenate-induced excitotoxic brain lesions. Such lesions are consistent with those observed in cerebral palsy. The present study builds on this murine model to assess the role of cyclooxygenase in interleukin-1-beta-induced brain toxicity. Pups pretreated with interleukin-1-beta developed greater ibotenate-induced brain damage than controls, an effect blocked by the co-administration of nimesulide (cyclooxygenase-2 inhibitor) or indomethacin (cyclooxygenase-1 and -2 inhibitor). Cyclooxygenase inhibitor administration prevented the interleukin-1-beta-induced increase in the production of brain prostaglandin E₂ (a cyclooxygenase metabolite) and changes in the expression of brain interleukin-6, interleukin-18, tumor necrosis factor-alpha, and brain-derived neurotrophic factor. It also stimulated the expression of brain interleukin-10. Our data suggest that the sensitizing effects of circulating inflammatory cytokines on the brain are mediated by the inducible isoform cyclooxygenase-2, which generates excess prostaglandin E₂. Some of these deleterious effects could involve an autocrine/paracrine loop leading to a disruption of the balance between pro- and anti-inflammatory cytokines in the brain.

Introduction

Despite a reduction in neonatal mortality and morbidity in the last 40 years, rates of cerebral palsy (CP) remain significant in Western countries (Hagberg et al., 1996, Himmelmann et al., 2005). Recently hypothesized etiologies of CP have gone beyond hypoxic–ischemic mechanisms to include multiple preconceptional and prenatal factors such as hypoxia/perfusion failure, genetic components, growth-factor deficiency, and maternal infection and inflammation leading to the production of excess cytokines (Nelson and Willoughby, 2000, Volpe, 2001, Dammann et al., 2002, Gressens et al., 2002).

The potential deleterious role of perinatal inflammation has been proposed for both preterm and full term neonates at risk for the development of brain lesions and CP (Murphy et al., 1995, Zupan et al., 1996, Nelson and Willoughby, 2000, Volpe, 2001, Dammann et al., 2002, Gressens et al., 2002). According to the mechanism hypothesized, in utero infection and/or inflammation induce a transplacental inflammatory response associated with the production of excess circulating cytokines capable of harming the developing brain. Findings from several studies support an association between maternal–fetal infection, circulating cytokines (such as interleukin or IL-1-beta, IL-6, and tumor necrosis factor or TNF-alpha), and periventricular white matter damage (PWMD) in preterm infants (Romero et al., 1990, Greig et al., 1993, Singh et al., 1996, Yoon et al., 1996, Yoon et al., 1997, Martinez et al., 1998). In addition, a striking association between increased levels of perinatal circulating cytokines, including IL-1-beta, IL-6, IL-8, IL-9, and TNF-alpha, and the subsequent occurrence of CP in full-term infants has been reported (Nelson et al., 1998).

Using a murine model of neonatal excitotoxic brain lesions based on the intracerebral administration of ibotenate, a glutamate analog acting on N-methyl-d-aspartate (NMDA) and metabotropic receptors, we have previously shown that pups pretreated with IL-1-beta, IL-6, or TNF-alpha develop significantly greater ibotenate-induced cortical and white matter damage than controls (Dommergues et al., 2000). The precise molecular mechanisms by which circulating mediators of inflammation have a deleterious effect on perinatal brain lesions remain a matter for debate (Hagberg and Mallard, 2005). Circulating cytokines do not seem to cross the intact blood–brain barrier (BBB). However, three alternative pathways have been proposed to link serum cytokine levels with brain lesion sizes. Firstly, circulating cytokines could alter the permeability of the BBB to inflammatory mediators and cells. Secondly, circulating cytokines could act directly on parts of the brain lacking the BBB such as the circumventricular organs, meninges and choroid plexus or, as demonstrated in the adult brain, indirectly through the activation of the vagal nerve. Thirdly, cytokine effects could be mediated by cyclooxygenase (Cox) located on the BBB. In particular, cytokines could activate the inducible isoform Cox-2 to enhance the local production of prostaglandin E₂ (PGE₂) that could have deleterious effects on the developing brain. Some of these deleterious effects could involve an autocrine/paracrine loop leading to the excess production of inflammatory cytokines by brain cells.

In the present study, using the murine model of neonatal excitotoxic brain lesions described above, we explored the mechanisms by which systemically injected inflammatory cytokines sensitize the developing brain. We investigated the impact of systemically administered IL-1-beta on the production of cytokines by brain cells, focusing on the potential role of Cox-2 in this process. To do this, we used indomethacin and nimesulide, two non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit Cox activity. Our data support the previously reported epidemiological association between high levels of circulating pro-inflammatory cytokines and an elevated risk of developing CP and/or PWMD.