title: Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.
abstract: BACKGROUND
  The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-).
  
  
  RESULTS
  In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release.
  
  
  CONCLUSIONS
  To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515, USA. amayer@midwestern.edu
authors.forename: A M
authors.initials: AM
authors.lastname: Mayer
authors.forename: M
authors.initials: M
authors.lastname: Hall
authors.forename: M J
authors.initials: MJ
authors.lastname: Fay
authors.forename: P
authors.initials: P
authors.lastname: Lamar
authors.forename: C
authors.initials: C
authors.lastname: Pearson
authors.forename: W C
authors.initials: WC
authors.lastname: Prozialeck
authors.forename: V K
authors.initials: VK
authors.lastname: Lehmann
authors.forename: P B
authors.initials: PB
authors.lastname: Jacobson
authors.forename: A M
authors.initials: AM
authors.lastname: Romanic
authors.forename: T
authors.initials: T
authors.lastname: Uz
authors.forename: H
authors.initials: H
authors.lastname: Manev
mesh_terms: D000818:Animals; D039481:CD11b Antigen / Q000032:analysis; D002470:Cell Survival / Q000187:drug effects; D002531:Cerebellum / Q000187:drug effects; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000633:toxicity*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008387:Marine Toxins / Q000633:toxicity; D013058:Mass Spectrometry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000032:analysis; D017470:Receptors, Glutamate / Q000032:analysis; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D039481:CD11b Antigen; D008070:Lipopolysaccharides; D008387:Marine Toxins; D012333:RNA, Messenger; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D014409:Tumor Necrosis Factor-alpha; C104724:glutamate receptor ionotropic, AMPA 4; D013481:Superoxides; D020780:Matrix Metalloproteinase 9; C012301:domoic acid; D007608:Kainic Acid