Expression of N-methyl D-aspartate receptor subunits in amoeboid microglia mediates production of nitric oxide via NF-κB signaling pathway and oligodendrocyte cell death in hypoxic postnatal rats

Glia. 2011 Apr;59(4):521-39. doi: 10.1002/glia.21121. Epub 2011 Jan 6.

Abstract

The present study was focused on identifying the expression of N-methyl D-aspartate receptor (NMDAR) subunits on activated microglia and to determine their role in the pathogenesis of periventricular white matter damage (PWMD) in neonatal rats following hypoxia. One day old wistar rats were subjected to hypoxia (5% O(2) ; 95% N(2) ) and the mRNA and protein expression of NMDAR subunits (NR1, NR2A-D, and NR3A) in the periventricular white matter (PWM) was determined at different time points (3,24 h, 3, 7, and 14 days) following hypoxic exposure. Immunoexpression of NR1 and NR2A-D was localized in amoeboid microglial cells (AMC) suggesting the presence of functional NMDARs in them. The expression of NMDAR in primary microglial cultures was ascertained by RT-PCR analysis and double immunofluorescence studies. The functionality of the microglial NMDAR in cultured microglial cells was examined by monitoring calcium movements in cells with fura-2. In primary microglial cultures, hypoxia induced the nuclear translocation of NF-κB which was suppressed by administration of MK801, an NMDAR antagonist. MK801 also down regulated the hypoxia-induced expression of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) production by microglia which may be mediated by the NF-κB signaling pathway. NO produced by microglia is known to cause death of oligodendrocytes in the developing PWM. In this connection, pharmacological agents such as MK801, BAY (NF-κB inhibitor), and 1400w (iNOS inhibitor) proved to be beneficial since they reduced the hypoxia-induced iNOS expression, NO production, and a corresponding reduction in the death of oligodendrocytes following hypoxia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidines / pharmacology
  • Animals
  • Animals, Newborn
  • Benzylamines / pharmacology
  • Blotting, Western
  • Calcium / metabolism
  • Cell Death / drug effects
  • Cell Death / physiology
  • Cells, Cultured
  • Dizocilpine Maleate / pharmacology
  • Excitatory Amino Acid Antagonists / pharmacology
  • Fluorescent Antibody Technique
  • Hypoxia / metabolism*
  • Hypoxia / pathology
  • Immunohistochemistry
  • In Situ Nick-End Labeling
  • Microglia / drug effects
  • Microglia / metabolism*
  • Microglia / pathology
  • NF-kappa B / metabolism*
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Oligodendroglia / drug effects
  • Oligodendroglia / metabolism*
  • Oligodendroglia / pathology
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Protein Transport / physiology
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Wistar
  • Receptors, N-Methyl-D-Aspartate / genetics
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*

Substances

  • Amidines
  • Benzylamines
  • Excitatory Amino Acid Antagonists
  • N-(3-(aminomethyl)benzyl)acetamidine
  • NF-kappa B
  • Protein Subunits
  • RNA, Messenger
  • Receptors, N-Methyl-D-Aspartate
  • Nitric Oxide
  • Dizocilpine Maleate
  • Nitric Oxide Synthase
  • Calcium