title: An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons.
abstract: The currently accepted scheme for reactive oxygen species production during ischemia/reperfusion injury is characterized by a deleterious mitochondria-derived burst of radical generation during reperfusion; however, recent examination of the penumbra suggests a central role for NADPH-oxidase (Nox)-mediated radical generation during the ischemic period. Therefore, we utilized a novel in vitro model of the penumbra to examine the free radical profile of ischemic murine hippocampal neurons using electron paramagnetic resonance spectroscopy, and also the role of Nox in this generation and in cell fate. We report that free radical production increased ~75% at 2 h of ischemia, and this increase was abolished by: (1) scavenging of extracellular free radicals with superoxide dismutase (SOD), (2) a general anion channel antagonist, or (3) the Nox inhibitor apocynin. Similarly, at 24 h of ischemia, [ATP] decreased >95% and vital dye uptake increased 6-fold relative to controls; whereas apocynin, the Cl(-) channel antagonist 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), or the free radical scavenger N-acetyl cysteine (NAC) each provided moderate neuroprotection, ameliorating 13-32% of [ATP]-depletion and 19-56% of vital dye uptake at 24 h. Our results support a cytotoxic role for Nox-mediated free radical production from penumbral neurons during the ischemic period.
authors.affiliation: Department of Pediatrics, Division of Respiratory Medicine, University of California San Diego, La Jolla, CA 92093, USA. mpamenter@ucsd.edu
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Pamenter
authors.forename: Sameh S
authors.initials: SS
authors.lastname: Ali
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Tang
authors.forename: J Cameron
authors.initials: JC
authors.lastname: Finley
authors.forename: Xiang Q
authors.initials: XQ
authors.lastname: Gu
authors.forename: Laura L
authors.initials: LL
authors.lastname: Dugan
authors.forename: Gabriel G
authors.initials: GG
authors.lastname: Haddad
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
chemical_list: D000098:Acetophenones; D017382:Reactive Oxygen Species; D013481:Superoxides; C056165:acetovanillone; D019255:NADPH Oxidases; D000111:Acetylcysteine