Seizure-induced alterations in NKCC1 and KCC2 protein levels.

<p>Using Western Blot, NKCC1 and KCC2 protein levels were assessed in cortical and hippocampal tissue obtained from P10 rats at 1, 12, 24, 48, and 168 hr after hypoxia-induced seizures (HS), and compared to age-matched littermate controls. Optical densities for NKCC1 and KCC2 were normalized to actin, averaged for each time point, and then the average expression in hypoxic animals was normalized to that in control animals. NKCC1 (A) and KCC2 (B) expression was plotted as a percent of control (dotted line). NKCC1 increased transiently at 24 hr post-HS (n = 8, 138±10.5% control, <i>p</i> = 0.005) in cortex, but showed no significant change in hippocampus. KCC2 expression increased early at 1 hr post-HS (n = 4, 140±9.6% control, <i>p</i> = 0.004) in hippocampus, with a later cortical increase 24 hr post-HS (n = 8, 81±6.7% control, <i>p</i> = 0.025). The normalized ratio of NKCC1 to KCC2 (C) was then calculated as a measure of changes in the relative expression of NKCC1 and KCC2 from normal expression patterns. In the cortex, the ratio of NKCC1 to KCC2 initially decreased 1 hr after seizures (n = 7, 0.66±0.09 fold, p = 0.003), and then increased at 12 and 24 hr (12 hr: n = 7, 1.71±0.27 fold, <i>p</i> = 0.046; 24 hr: n = 8, 1.83±0.26 fold, <i>p</i> = 0.008), as compared to controls. In the hippocampus, NKCC1/KCC2 was significantly higher at 12 hr (n = 8, 1.39±0.09 fold, p = 0.016), as compared to controls. (D) Representative western blot images for NKCC1, KCC2, and actin protein levels in cortex (top) and hippocampus (bottom). C = control; HS = hypoxic seizures. Mean ± SEM. Error bars indicate SEM.*<i>p</i><0.05, **<i>p</i><0.01.