Elsevier

Journal of Neuroimmunology

Volume 109, Issue 2, 22 September 2000, Pages 181-187
Journal of Neuroimmunology

Role of NMDA receptor signaling in the regulation of inflammatory gene expression after focal brain ischemia

https://doi.org/10.1016/S0165-5728(00)00317-9Get rights and content

Abstract

Inflammatory mediators are involved in the pathogenesis of focal ischemic brain damage. In this study we used quantitative reverse transcriptase–polymerase chain reaction to analyze the spatiotemporal pattern of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) expression in focal ischemia of the rat brain. Focal ischemia of the rat parietal cortex was induced noninvasively by photothrombosis of cortical microvessels. In a proportion of the animals NMDA receptor signaling was blocked by the noncompetitive receptor antagonist MK-801. Within 4 h after ischemia we found induction of TNF-α and IL-1β mRNA not only in the infarcts but also in all representative tissue samples removed from noninfarcted frontal, lateral, and occipital cortex of the ipsilateral, but not contralateral hemisphere. Contrastingly, the expression of iNOS mRNA remained restricted to the evolving infarcts. Pretreatment with MK-801 strongly inhibited remote cytokine expression (mean reduction by 80% relative to vehicle treated animals at 4 h; P<0.001) whereas in the lesions only partial reductions in the expression of IL-1β and iNOS mRNA were found. Our data for the first time demonstrate remote cytokine induction following focal brain ischemia and suggest that NMDA receptor-mediated signaling can activate inflammatory gene expression independently from the occurrence of neuronal cell death.

Introduction

Cytokines and inflammation in the central nervous system (CNS) have been primarily studied in the context of autoimmune and infectious diseases. However, ‘nonimmune’ lesions such as focal brain ischemia likewise trigger a strong inflammatory response that involves the recruitment of macrophages and T cells as well as activation of local glia (Arvin et al., 1996, Stoll et al., 1998). The role of cytokines for lesion development is incompletely understood. At least in some in vitro paradigms proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) exerted neurotoxic effects that depended on the concomitant induction of the inducible nitric oxide synthase (iNOS) in activated glial cells (Hewett et al., 1994, Chao et al., 1995). In ischemic brain lesions in vivo, resident glia (Bidmon et al., 1998a, Loihl et al., 1999) as well as infiltrating leukocytes and blood vessels (Iadecola et al., 1995, Galea et al., 1998) have been suggested as potential sources of iNOS activity. In line with a role of iNOS in the exacerbation of ischemic brain damage targeting of the iNOS gene in mice significantly reduced the delayed expansion of ischemic brain lesions induced by permanent occlusion of the middle cerebral artery (Iadecola et al., 1997). TNF-α moreover increases the adhesiveness of the microvascular endothelium for circulating polymorphonuclear cells that exacerbate brain damage by plugging of capillaries during subsequent reperfusion of the ischemic tissue (Zhang et al., 1995, Connolly et al., 1996). In some studies, early neutralization of TNF-α and IL-1β decreased the lesion size after permanent middle cerebral artery occlusion in rats (Relton and Rothwell, 1992, Barone et al., 1997). Contrastingly, findings in TNF receptor-deficient mice suggested a protective role of endogenously produced TNF-α in focal ischemia (Bruce et al., 1996) that is supported by in vitro studies showing a decrease of excitotoxic neuronal death after pretreatment with inflammatory cytokines (Cheng et al., 1994, Strijbos and Rothwell, 1995). Similarly, pretreatment of animals with either TNF-α or IL-1β decreased neuronal damage upon subsequent brain ischemia (Ohtsuki et al., 1996, Nawashiro et al., 1997).

Cellular responses to focal ischemia are not restricted to the ischemic lesion but likewise involve nonischemic cortical areas with activation of glial cells and neuronal expression of the inflammatory enzyme cyclooxygenase-2 (Schroeter et al., 1995, Schroeter et al., 1999, Miettinen et al., 1997). Most of these changes are observed in the ipsilateral but not contralateral hemisphere and can be blocked by the noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 suggesting that they are mainly induced by infarct-triggered cortical spreading depression. Functionally, spreading depression exacerbates ischemic brain damage in the penumbra zone of the lesion (Hossmann, 1996) but induces protective effects in nonischemic ipsilateral cortical areas (Kobayashi et al., 1995). To clarify the possible contribution of inflammation to remote effects of focal brain ischemia we have analyzed the spatiotemporal pattern of TNF-α, IL-1β, and iNOS expression in rat models of focal brain ischemia and addressed the role of NMDA receptor signaling in the induction of postischemic inflammatory gene expression.

Section snippets

Animal experiments

Focal cortical infarctions were induced in adult male Wistar rats by either permanent occlusion of the middle cerebral artery (MCAO) or photothrombosis of cortical microvessels under inhalation anesthesia with halothane [1.3% in an O2/N2 (1:2) mixture] as described in detail elsewhere (Schroeter et al., 1994, Jander et al., 1995). All animal experiments were performed in accordance with institutional guidelines. In the MCAO model, infarcted and noninfarcted contralateral cortex was analyzed at

Results

In the MCAO model of focal ischemia large infarcts are produced making a clear differentiation between ischemic and nonischemic tissue difficult. For the reliable analysis of cytokine induction in noninfarcted ipsilateral cortex we therefore chose the photothrombosis model in which small, sharply demarcated lesions are induced noninvasively. In initial experiments using semiquantitative RT-PCR we compared the inflammatory responses within photochemically induced ischemic lesions to those

Discussion

As main finding our study shows that the expression of TNF-α and IL-1β mRNA after focal brain ischemia was not restricted to the ischemic lesions but likewise occurred in all representative tissue samples removed from non-infarcted frontal, lateral, and occipital cortex of the ipsilateral, but not contralateral hemisphere. In contrast to both cytokines iNOS mRNA remained restricted to the ischemic lesion at all time points. Cytokine induction in the nonischemic ipsilateral cortex was almost

Acknowledgements

We thank B. Blomenkamp for excellent technical assistance, B. Ehle for statistical analysis, and U. Pippirs for help with automated DNA sequencing. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 194, B6). GS holds a Hermann- and Lilly-Schilling professorship.

References (51)

  • A.K. Loihl et al.

    Expression of nitric oxide synthase (NOS)-2 following permanent focal ischemia and the role of nitric oxide in infarct generation in male, female and NOS-2 gene-deficient mice

    Brain Res.

    (1999)
  • A.M. Marini et al.

    Activity-dependent release of brain-derived neurotrophic factor underlies the neuroprotective effect of N-methyl-d-aspartate

    J. Biol. Chem.

    (1998)
  • J.K. Relton et al.

    Interleukin-1 receptor antagonist inhibits ischaemic and excitotoxic neuronal damage in the rat

    Brain Res. Bull.

    (1992)
  • M. Schroeter et al.

    Local immune responses in the rat cerebral cortex after middle cerebral artery occlusion

    J. Neuroimmunol.

    (1994)
  • M. Schroeter et al.

    Heterogeneity of the microglial response in photochemically induced focal ischemia of the rat cerebral cortex

    Neuroscience

    (1999)
  • G. Stoll et al.

    Inflammation and glial responses in ischemic brain lesions

    Prog. Neurobiol.

    (1998)
  • F.C. Barone et al.

    Tumor necrosis factor-α: a mediator of focal ischemic brain injury

    Stroke

    (1997)
  • H.J. Bidmon et al.

    Transient changes in the presence of nitric oxide synthases and nitrotyrosine immunoreactivity after focal cortical lesions

    Neuroscience

    (1998)
  • H.J. Bidmon et al.

    Structural alterations and changes in cytoskeletal proteins and proteoglycans after focal cortical ischemia

    Neuroscience

    (1998)
  • G.I. Botchkina et al.

    Expression of TNF and TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia

    Mol. Med.

    (1997)
  • J.S. Braun et al.

    Spatiotemporal relationship of apoptotic cell death to lymphomonocytic infiltration in photochemically induced focal ischemia of the rat cerebral cortex

    Acta Neuropathol. (Berl.)

    (1996)
  • A.J. Bruce et al.

    Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors

    Nat. Med.

    (1996)
  • E.S. Connolly et al.

    Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion: role of neutrophil adhesion in the pathogenesis of stroke

    J. Clin. Invest.

    (1996)
  • W.D. Dietrich et al.

    Photothrombotic infarction triggers multiple episodes of cortical spreading depression in distant brain regions

    J. Cereb. Blood Flow Metab.

    (1994)
  • C. Forster et al.

    Inducible nitric oxide synthase expression in human cerebral infarcts

    Acta Neuropathol. (Berl.)

    (1999)
  • Cited by (114)

    • The cerebral endothelial cell as a key regulator of inflammatory processes in sterile inflammation

      2019, Journal of Neuroimmunology
      Citation Excerpt :

      Contrarily the ability of locally loosening the BBB can offer possibilities of more effective administration of drugs to certain brain regions (Vazana et al., 2016). Glutamate and the corresponding NMDA receptor have been found to play a role in BBB opening in pathological conditions with inhibition of NMDA receptors ameliorating BBB leakage in murine epilepsy and stroke, as well as dampening expression of inflammatory cytokines such as TNF-α (Jander et al., 2000). Mechanistically, stimulation of NMDA receptors has shown to elevate intracellular calcium and nitrogen monoxide levels and lead to redistribution and phosphorylation of occludin, reducing junctional integrity (Andras et al., 2007; De Bock et al., 2013).

    View all citing articles on Scopus
    View full text