Fig. 1

Hippocampal nNOS immunoreactivity in normoxic and hypobaric hypoxic rats pretreated with normal saline or melatonin, and sacrificed at 0 h, 1 and 3 days of reoxygenation. Light photomicrographs (a) show that light-stained nNOS immunoreactivity (arrows) is mainly found in pyramidal cells scattered throughout the CA1 region in normoxic rats (A, B). nNOS immunoreactivities are drastically increased at 0 h, 1 and 3 days after hypoxic exposure (C, E, G). In rats receiving melatonin pretreatment and subjected to 0 h- (D), 1- (F) and 3- (H) days hypoxic exposure, hippocampal nNOS immunoreactivity is significantly reduced. The inserts indicate nNOS(+) neurons of higher magnified in each representative figure. Scale bar 50 μm for all figures, insert 100 μm. Histograms showing the mean optical density of nNOS(+) neurons (b) and expression of total nNOS protein (c) quantified by immunoblots in the hippocampus of rats treated with hypoxia alone (black column) and melatonin pretreated (white column) and sacrificed at 0 h, 1 and 3 days of reoxygenation. Note that in hypoxic rats, the staining intensity and the levels of total protein of nNOS are drastically increased. In rats received melatonin, the nNOS staining and protein levels are successfully decreased in the hippocampus at the beginning and 1 day of reoxygenation. Dashed line shows the baseline controls are set as 100 % (saline or melatonin treatment under normoxic condition). The levels of β-actin are as a loading control (c). *P < 0.05 (Student’s t test) when compared with values (expressed as mean ± SEM) of rats treated only with hypoxia at the same survival time point