Glycogen Synthase Kinase-3 and TBI Figure 2. Systemic lithium administration inhibits hippocampal GSK-3 activity. A) Representative western blots showing the dose response of different amounts of lithium (injected daily for 5 days) on GSK-3ß phosphorylation. The summary data shows that all doses of lithium were found to increase GSK-3 phosphorylation. B) Representative western blots and summary data showing that the different doses of lithium had no effect on the total levels of GSK-3ß. C) Representative western blots and summary data showing that lithium administration causes a significant accumulation of ß-Catenin, with maximal levels seen at the 1.0 mEq dose. D) Representative western blots and summary data showing that a single dose of 1.0 mEq/Kg lithium has no effect on the phosphorylation or total levels of GSK-3ß, nor on ß-Catenin accumulation. Data are presented as the mean 6 SEM. *, P#0.05 by one-way ANOVA compared to vehicle (0 dose) injected animals. n = 3/group. doi:10.1371/journal.pone.0024648.g002 Figure 3. Post-TBI lithium administration has no effect on motor function, but exacerbates post-injury weight loss. A) Schematic of the injection and behavioral testing paradigm. Injured rats receiving 1 mEq/kg lithium (n = 10) performed similarly to vehicle- injected animals (n = 10) when tested for their B) balance beam performance, and C) ipsilateral and D) contralateral foot faults. E) Lithium exacerbated post-injury weight loss. Data are presented as the mean 6 SEM. ?, significant difference by repeated measures two- way ANOVA. *, P#0.05. doi:10.1371/journal.pone.0024648.g003 improved in animals treated with lithium, as evidenced by reduced latency to cross the original platform location (Student’s t-test p = 0.034) and more number of platform crossings (Student’s t-test p = 0.004) (Figure 4D) in a probe trial given 30 min post-training. The improved water maze performance we observed as a result of lithium treatment suggests that this drug may offer neuronal protection to hippocampal neurons after TBI. When the brains of the animals used for behavioral testing were examined for hippocampal neuron loss, an apparent preservation of ipsilateral CA3 pyramidal neurons (Figures 5A) was observed. To quantify the number of ipsilateral CA3 neurons, sections spanning the dorsal hippocampus from representative vehicle (n = 5)- and lithium (n = 5)-treated animals were immunoreacted for NeuN. Immunoreactivity was detected using a horseradish peroxidase- coupled secondary antibody, and then visualized using diamino- benzidine (Figure 5B). Stereological cell counts of CA3 pyramidal neurons were performed using the optical dissector probe of Stereo Investigator. These counts revealed that animals treated with lithium had significantly more ipsilateral CA3 pyramidal neurons than those counted in vehicle-injected animals (Figure 5C). Cortical contusion volume, however, was not significantly different between the two groups (Figure 5D). The GSK-3 inhibitor SB-216763 improves motor function following TBI As our results with lithium suggested that GSK-3 inhibition may offer neuroprotection, we next tested if a GSK-3-specific inhibitor could mimic the influences of lithium. SB-216763 has been shown to be a potent inhibitor of GSK-3, while having no significant influence on the activity of other kinases [26]. To test the efficacy of the drug, rats were i.p. injected with either 2.5 or 5.0 mg/kg SB-216763 (n = 3/group), euthanized 1 hr later, and the hippo- campus dissected for the preparation of protein extracts. These doses were chosen based on previous studies indicating that SB- 216763 can reduce D1 agonist-induced hyperactivity [28]. Representative western blots are shown in Figure 6A. Consistent PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24648