on April 16, 2009
Published online before print April 16, 2009, doi: 10.1161/STROKEAHA.108.544759
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Submitted on December 9, 2008
From Pediatrics (M.T.Z., F.H., G.V., E.F.L.), Maria Fareri Children's Hospital, New York Medical College, Valhalla, NY; and Physiology (A.C., N.L., Z.U.) and Pediatrics, Cell Biology and Anatomy (P.B.), New York Medical College, Valhalla, NY. * To whom correspondence should be addressed. E-mail: Pballabh{at}msn.com.
Background and Purpose—Germinal matrix hemorrhage–intraventricular hemorrhage is the most common neurological problem of premature infants. Despite this, mechanisms of brain injury from intraventricular hemorrhage are elusive. We hypothesized that germinal matrix hemorrhage–intraventricular hemorrhage, by induction of NAD(P)H oxidases, might cause oxidative/nitrosative stress contributing to brain injuries and that NAD(P)H oxidase inhibition could offer neuroprotection. Methods—To test this hypothesis, we exploited our rabbit pup model of glycerol-induced germinal matrix hemorrhage–intraventricular hemorrhage. We delivered rabbit pups prematurely (E29) by cesarean section and administered intraperitoneal glycerol at 2 hours postnatal age. Free-radical adducts, including nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine as well as O2.- and H2O2 levels were measured in the forebrain. To determine the source of free-radical generation, we used inhibitors for NAD(P)H oxidase (apocynin), xanthine oxidase (allopurinol), cyclo-oxygenase-2 (indomethacin), or nitric oxide synthases (L-NAME). Intraventricular hemorrhage pups were treated with apocynin and cell death was compared between apocynin-treated and vehicle-treated pups. Results—Nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine levels were higher in pups with intraventricular hemorrhage than controls. Likewise, O2.- and H2O2 levels were significantly greater in both the periventricular area and cerebral cortex of pups with intraventricular hemorrhage than controls. In pups with intraventricular hemorrhage, reactive oxygen species production was more in the periventricular area than in the cortex. Apocynin, but not allopurinol, indomethacin, or nitric oxide synthases, inhibited reactive oxygen species generation. Importantly, apocynin reduced cell death in pups with intraventricular hemorrhage. Conclusion—Activation of NAD(P)H oxidase was the predominant mechanism of free-radical generation in pups with intraventricular hemorrhage. NAD(P)H oxidase inhibition by apocynin might suppress reactive oxygen species production and confer neuroprotection in premature infants with intraventricular hemorrhage.
Revised on January 16, 2009
Accepted on February 2, 2009
Oxidative–Nitrosative Stress in a Rabbit Pup Model of Germinal Matrix Hemorrhage. Role of NAD(P)H Oxidase
Muhammad T. Zia MD;
Key words: apocynin • germinal matrix hemorrhage–intraventricular hemorrhage • NADPH oxidase • nitrotyrosine • oxidative stress • reactive oxygen species • reactive nitrogen species • superoxide