Oxidative-nitrosative stress in a rabbit pup model of germinal matrix hemorrhage: role of NAD(P)H oxidase.
OBJECTIVE
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 O(2)(.-) and H(2)O(2) 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, O(2)(.-) and H(2)O(2) 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.


CONCLUSIONS
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.
Pediatrics, Maria Fareri Children's Hospital at Westchester Medical Center, Valhalla, NY 10595, USA.
Muhammad T
MT
Zia
Anna
A
Csiszar
Nazar
N
Labinskyy
Furong
F
Hu
Govindaiah
G
Vinukonda
Edmund F
EF
LaGamma
Zoltan
Z
Ungvari
Praveen
P
Ballabh
D000098:Acetophenones / Q000494:pharmacology; D000166:Acridines; D000818:Animals; D015153:Blotting, Western; D002543:Cerebral Hemorrhage / Q000000981:diagnostic imaging / Q000201:enzymology / Q000378:metabolism*; D002552:Cerebral Ventricles / Q000473:pathology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004996:Ethidium; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D049449:Luminescence; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D018696:Neuroprotective Agents; D018384:Oxidative Stress / Q000502:physiology*; D011817:Rabbits; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014463:Ultrasonography
D000098:Acetophenones; D000166:Acridines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018696:Neuroprotective Agents; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C033472:10,10'-dimethyl-9,9'-biacridinium; C056165:acetovanillone; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D004996:Ethidium