iNOS-expression in immature microglia

HYPEROXIA INDUCES MICROGLIAL EXPRESSION OF INOS IN THE IMMATURE RAT BRAIN

Hoehn T, Felderhoff-Mueser U, Sifringer M, Koehne P, Bittigau P, Buhrer C

Background:

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.

Aim:

To investigate the effect of hyperoxia on the amount and topographic distribution of iNOS-expression (inducible nitric oxide synthase) in the immature rat brain. Furthermore to localize hyperoxia-induced formation of peroxynitrite as a potential marker of cellular damage to immature cerebral structures.

Methods:

7 day old Wistar rat pups were exposed to >80% oxygen for 24 hours 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.

Results:

Total brain iNOS mRNA was upregulated, demonstrating a peak at 6 hours following the onset of hyperoxia. There was a threefold increase in iNOS protein in retrosplenial cortex and hippocampus when compared to control animals. 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. There was no positive staining for iNOS or nitrotyrosine in control animals.

Conclusions:

Hyperoxia causes iNOS mRNA and protein upregulation in microglial cells of the immature rat brain. Positive neuronal nitrotyrosine staining indicates formation of peroxynitrite with potential deleterious effects for immature cellular structures in the neonatal brain.