This Article Full Text Full Text (PDF) All Versions of this Article: 40/6/2191    most recent STROKEAHA.108.544759v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Zia, M. T. Articles by Ballabh, P. PubMed PubMed Citation Articles by Zia, M. T. Articles by Ballabh, P. Related Collections Acute Cerebral Hemorrhage Neuroprotectors

(Stroke. 2009;40:2191.)
© 2009 American Heart Association, Inc.

Original Contributions

Oxidative-Nitrosative Stress in a Rabbit Pup Model of Germinal Matrix Hemorrhage

Role of NAD(P)H Oxidase

Muhammad T. Zia, MD; Anna Csiszar, MD, PhD; Nazar Labinskyy, MD; Furong Hu, BA; Govindaiah Vinukonda, PhD; Edmund F. LaGamma, MD; Zoltan Ungvari, MD, PhD Praveen Ballabh, MD

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.

Correspondence to Praveen Ballabh, MD, Regional Neonatal Center, 2nd Floor, Maria Fareri Children’s Hospital at Westchester Medical Center, Valhalla, NY 10595. 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.

Key Words: apocynin • germinal matrix hemorrhage-intraventricular hemorrhage • NADPH oxidase • nitrotyrosine • oxidative stress • reactive oxygen species • reactive nitrogen species • superoxide