Neuropediatrics 2006; 37 - P36
DOI: 10.1055/s-2006-974047

Pharmacological inhibition of NADPH oxidase does not reduce excitotoxic brain lesion in newborn mice

E Griesmaier 1, E Kapeller 1, M Urbanek 1, C Humpel 2, G Wenning 3, G Simbruner 1, P Gressens 4, M Keller 1
  • 1Medical University Innsbruck, Department of Pediatrics, Neonatology, Innsbruck, Austria
  • 2Medical University Innsbruck, Department of Psychiatry, Innsbruck, Austria
  • 3Medical University Innsbruck, Department of Neurology, Innsbruck, Austria
  • 4INSERM, Hopital Robert Debre, Paris, France

Aims: In premature infants, excitotoxicity, inflammation and oxidative stress are factors contributing to perinatal brain injury. Excitotoxic brain injury is also mediated by activation of microglial cells. These cells contain the enzyme NADPH oxidase which generates reactive oxygen species (ROS). The role of NADPH oxidase in perinatal brain injury is unknown.

Hypothesis: We hypothesize

  • excitotoxicity induces free radical formation by NADPH oxidase in microglial cells in vivo,

  • inhibition of NADPH oxidase by local (peptide gp91ds-tat) or systemic administration (apocynin (apo)) reduces excitotoxic brain injury in newborn mice in vivo.

Methods: In vitro: ROS formation on isolated microglial cells was measured by the DCF method with and without inhibition of NADPH oxidase.

In vivo: Ibotenic acid, 10µg (ibo) was injected into the right hemisphere (i.c.) of 5 day old mice to create white (WM) and gray matter lesions mimicking human perinatal brain damage. In the first track animals were randomized into two groups:

  • Ibo + scrambled–tat (1µM) i.c.

  • Ibo + gp91ds-tat (1µM) i.c..

In the second track animals were randomized into 4 groups:

  • Ibo i.c.+ vehicle intraperitoneal (i.p.), once,

  • Ibo i.c.+ apo (4mg/kg) i.p., once 1hr after injury,

  • Ibo i.c.+ apo i.p.12hrs before the insult,

  • ibo i.c.+ apo i.p. repetitively every 24hrs for 5 days, starting 1hr after i.c. injection. Lesion size, the number of apoptotic cells, oxidative stress (HNE staining) were determined at 4, 24 and 120hrs after injury.

Results: In vitro the ibotenate-induced free radical formation was blocked by gp91ds-tat. In vivo excitotoxicity induced oxidative stress at the lesion site. Neither the local nor systemic inhibition of NADPH oxidase reduced perinatal brain injury. On the contrary apo significantly increased lesion size in WM from 480±50 to 731±44µm.

Conclusion: Although effective in attenuating free radical formation in vitro, administration of NADPH oxidase inhibiting drugs were not neuroprotective. A therapeutical use of NADPH oxidase inhibiting drugs cannot be recommended.