Relative hyperoxia is a condition frequently encountered in premature infants, either spontaneously or during treatment in the Neonatal Intensive Care Unit. The effects of high inspiratory oxygen concentrations on immature brain cells and their signaling cascades are largely unknown. The aim of the study was to investigate the effect of hyperoxia on the amount and topographic distribution of iNOS-expression (inducible nitric oxide synthase) in the immature rat brain, and to localize hyperoxia-induced formation of peroxynitrite as a potential marker of cellular damage to immature cerebral structures. Seven-day-old Wistar rat pups were exposed to Ͼ80% oxygen for 24 h and were then transcardially perfused. Following paraformaldehyde fixation, brains were paraffin-embedded and immunohistochemically stained for iNOS and nitrotyrosine. iNOS protein was quantified by Western blot; iNOS mRNA expression was studied by RT-PCR. Total brain iNOS mRNA was up-regulated, demonstrating a peak at 6 h following the onset of hyperoxia. Immunohistochemical staining was predominantly observed in microglial cells of hippocampus and frontal cortex with some iNOS reactivity in endothelial and perivascular cells. Nitrotyrosine staining was positive in apical dendrites of neurons in the frontal cortex. The role of NO has been subject of numerous investigations during recent years. This applies to its function as a gaseous messenger molecule and to its vasodilative properties. NO is released by hydroxylation of nitrogen at the guanidino group of L-arginine, a reaction which is catalyzed by the enzyme NO synthase (NOS). At least three isoforms of NOS are known at the moment, two constitutive forms (endothelial NOS and neuronal NOS) and the inducible form (iNOS). The latter is induced by bacteria and cytokines. NO concentrations produced by iNOS are much higher than those generated by constitutive NOS (1), and have been shown to exert antimicrobial effects against some bacterial pathogens in vitro (2).As soon as NO is generated in the target cell (cancer cell, parasite), binding at copper and iron containing proteins takes place (3). As a result copper and iron are released and, in combination with oxygen, toxic radicals are formed. This is referred to as "massive oxidative injury" and requires the expression of iNOS leading to the production of huge amounts of NO, whereas low levels of NO synthesized by the constitutive forms of NOS (eNOS, nNOS) act as a signal molecule (4). Thus, the concentration of NO determines its biologic effect upon neighboring cells and tissues. The reaction products of nitrogen and oxygen include the strong oxidant peroxyni-

3D imaging of the ACL footprints reveals a distinct difference in insertion site morphology and fiber bundle orientation between the femoral and tibial footprint. This questions the concept of strict anatomical separation of the ACL into an anteromedial and posterolateral bundle.