Elsevier

Neuroscience

Volume 136, Issue 3, 2005, Pages 615-623
Neuroscience

Microglia response and P2 receptor participation in oxygen/glucose deprivation-induced cortical damage

https://doi.org/10.1016/j.neuroscience.2005.04.038Get rights and content

Abstract

In the present work, we used a unique cortical/striatal/subventricular zone organotypic model in order to analyze the role of resident microglia in oxygen/glucose deprivation and to check the presence and modulation of several P2 receptors in the cortex. Immunofluorescence with the microglial marker OX42 and pharmacological experiments with indomethacin indicate that activation and recruitment of microglia after the insult is linked to cellular loss, mainly in the cortex. The confocal analysis with OX42 shows that, among the P2 receptors tested, P2X4, and P2X7 are expressed on microglia, while P2X1 and P2Y1-2-12, although present in the slices, did not co-localize, whereas P2X6 is not detected. The upregulation of P2X4 and P2X7 on microglia and the toxic effect that different P2 agonists exert on cortical slices during oxygen/glucose deprivation indicate that a purinergic mechanism is related to the microglia activity; the protective effect of the P2 antagonist TNP-ATP is also described. In order to better understand the relationship between P2 receptors and OGD-activated microglia, we induced oxygen/glucose deprivation in co-cultures of organotypic slices and N9 microglia cell line. The presence of the N9 (which expresses P2X4 and P2X7 protein) in the cultures increases the damage in the cortex by 40% and the use of P2 antagonist PPADS reduced the cell damage due to the N9 activation. Our results show that microglia recruitment after a metabolic impairment is associated with cellular loss and that P2X4 and P2X7, are involved in microglia activity. The neuroprotective action exerted by TNP-ATP and PPADS and the possible use of purinergic antagonist in the pharmacological treatment of oxygen/glucose deprivation is also addressed.

Section snippets

Organotypic cortex–striatum–SVZ cultures

Cultures were prepared from cerebral coronal sections (400μm) of Wistar rat pups (2–3 days old) using a modification of the method by Plenz and Kitai (1996). Slices were dissected as shown in Fig. 1A in order to maintain the connection between the SVZ and corpus callosum. Slices were plated on Millicell CM culture inserts (Millipore, Schwalbach, Germany) and maintained in 75% HME 03 (Cell Concept, Berlin, Germany), 2mM l-glutamine (Biochrom, Berlin, Germany), 25% horse serum (Gibco, Eggenstein,

OGD effects on cortex/striatum/SVZ organotypic cultures

The organotypic slices were prepared as shown in Fig. 1A so that, the structures and relationship between primary motor–sensory cortex/caudate putamen/SVZ were preserved. The cultures were maintained for 40 min in OGD and cellular loss was evaluated 24 h later by propidium iodide incorporation (Fig. 1B). The main part of the damage was observed in the cortex, highlighting a marked cell death in the region likely corresponding to the cell layer IV (Fig. 1B, arrow), and to a lesser extent in the

Discussion

In this work we adopted the organotypic cultures to study the mechanisms of OGD-induced cell death. These cultures possess different advantages with respect to the in vitro dissociated cultures and the in vivo animal models. In fact, they preserve the organotypic network, tissue-specific transport, ion diffusion systems and neuron–glia interaction.

In particular, the use of the organotypic slice results in an efficient model to study the role of microglia reaction after an insult. They fail to

Acknowledgments

We thank Dr. C. Volonté (Santa Lucia Foundation, Rome) for providing us the P2 antibodies and Prof. O. Ullrich and his laboratory (Otto von Guericke University, Magdeburg) for the assistance with Western blots. N9 cell line was provided by Dr. P. Lorenz (Otto von Guericke University, Magdeburg). Work supported by Marie Curie development host fellowship, program contract number: MCFH-2001-00639.

References (47)

  • L. Lagunas et al.

    Indomethacin and ibuprofen induce Hsc70 nuclear localization and activation of the heat shock response in HeLa cells

    Biochem Biophys Res Commun

    (2004)
  • M. Minami et al.

    Chemokines and their receptors in the brainpathophysiological roles in ischemic brain injury

    Life Sci

    (2003)
  • K. Nakajima et al.

    Functional roles of microglia in the brain

    Neurosci Res

    (1993)
  • L.K. Parvathenani et al.

    P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer’s disease

    J Biol Chem

    (2003)
  • D. Plenz et al.

    Organotypic cortex-striatum-mesencephalon culturesthe nigrostriatal pathway

    Neurosci Lett

    (1996)
  • M. Schilling et al.

    Microglial activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemiaa study in green fluorescent protein transgenic bone marrow chimeric mice

    Exp Neurol

    (2003)
  • T. Suzuki et al.

    Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia

    J Neurosci

    (2004)
  • M. Yamada et al.

    Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death

    Brain Res

    (1994)
  • D. Yeung et al.

    Enhanced expression of the P2X4 receptor in Duchenne muscular dystrophy correlates with macrophage invasion

    Neurobiol Dis

    (2004)
  • F. Agasse et al.

    Neurogenic and intact or apoptotic non-neurogenic areas of adult brain release diffusible molecules that differentially modulate the development of subventricular zone cell cultures

    Eur J Neurosci

    (2004)
  • C. Boucsein et al.

    Purinergic receptors on microglial cellsfunctional expression in acute brain slices and modulation of microglial activation in vitro

    Eur J Neurosci

    (2003)
  • F. Cavaliere et al.

    Synaptic P2X7 and oxygen/glucose deprivation in organotypic hippocampal cultures

    J Cereb Blood Flow Metab

    (2004)
  • S.B. Corradin et al.

    Inducible nitric oxide synthase activity of cloned murine microglial cells

    Glia

    (1993)

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