Abstract 403

Previous studies have shown that inducible, calcium-independent isoform of nitric oxide synthase (iNOS) contributes significantly to hypoxic/ischemic neurotoxicity through the production of nitric oxide (NO). The present study aims to investigate the localization and temporal profile of iNOS expression after cerebral hypoxia in the hippocampus of the guinea pig fetus. Studies were performed on 15 pregnant guinea pigs divided into normoxic (Nx) and hypoxic groups. Hypoxia was induced by exposing the mothers to an FiO2 of 0.07 for 1 hour. Fetal brains were removed either immediately after the end of hypoxia (Hx0, n=2), or following 24hr (Hx24h, n=4) or 72hr (Hx72h, n=5) survival. Four brains from normoxic full-terms fetuses (Nx) served as controls. Brain tissue was fixed by impression in 10% phosphate-buffered formalin and embedded in paraffin. Immunohistochemistry was performed according to the avidin-biotin complex method, using an affinity-purified polyclonal antibody to iNOS (Transduction Laboratories). Mid-sagittal sections were analyzed and 400-600 neuronal cells were counted per each animal in dentate gyrus, CA1 and CA3 sectors of the hippocampal formation. The results were expressed as percentage of the total number of counted cells. In Nx fetuses no significant iNOS-like immunoreactivity is detected. By contrast, a marked increase of iNOS-like immunoreactivity in present in Hx0hr fetuses. It involves predominantly pyramidal neurons of the CA3 sector (69% ± 39% in Hx0hr vs 0.9% in Nx) and to a lesser degree CA1 sector and granule neurons of the dentate gyrus. In Hx24hr and Hx72h fetuses there is a substantial decrease in the distribution of iNOS-like immunoreactivity with only scattered neurons in the CA3 exhibiting staining (11% ± 19% in Hx24hr, 10% ± 13% in Hx72hr). Thus, compared to Nx controls, Hx24hr and Hx72hr fetuses, a significant increase in neuronal iNOS-like immunoreactivity is present only in the CA3 subfield of Hx0 fetuses (p=0.016 vs Nx; p=0.059 vs Hx24h; and p=0.021 vs Hx72r). Our data indicate a transient and acute increase of iNOS-like immunoreactivity in selective hippocampal neurons (CA3) during hypoxia of the guinea pig fetus at term. We speculate that transient increase in iNOS expression in neurons will lead to increased NO production and may potentiate NMDA receptor-mediated neuronal injury.