Glycogen Synthase Kinase-3 and TBI Figure 4. Post-injury administration of lithium chloride im- proves cognitive function following TBI. Rats treated with 1 mEq/ kg lithium (n = 10) had improved spatial learning and memory as indicated by A) decreased latencies to the platform during training (?, significant difference by repeated measures two-way ANOVA), and B) decreased latency to the first platform crossing and increased number of platform crossings during a probe trial given 24 hr after training (*, p#0.05 by Student’s t-test). C) Animals receiving lithium spent significantly longer time in the target quadrant (I) compared to the other quadrants (II, III, IV), suggesting improved localization (*, p#0.05 by repeated measures one-way ANOVA). D) A probe trial given 30 min after training showed that the lithium-infused animals also had significantly shorter latencies and more crossings of the platform location compared to vehicle-infused animals (*, p#0.05 by Student’s t- test). Veh: vehicle; Li: lithium. Data are presented as the mean 6 SEM. doi:10.1371/journal.pone.0024648.g004 with previous reports, Figure 6B shows that SB-216763 increases the phosphorylation of GSK-3b on Ser9 (one-way ANOVA: F(2,6) = 8.833, p = 0.016), suggesting decreased GSK-3 activity. Associated with this was a significant decrease in the phosphor- ylation of LRP6 on Ser1490 (one-way ANOVA: F(2,6) = 5.891, p = 0.038; Figure 6C) and a trend towards decreased ß-Catenin phosphorylation on Ser33/37 (one-way ANOVA: F(2,6) = 4.697, p = 0.059; Figure 2D). No significant influence of SB-216763 was observed on the phosphorylation/activity of Akt (one-way ANOVA: F(2,6) = 0.381, p = 0.698; Figure 6E) or ERK1/2 (one- way ANOVA: F(2,6) = 0.865, p = 0.468; Figure 2F), nor on the total levels of any of the targets interrogated. In order to determine the influence of SB-216763 on TBI outcome, rats were injured, then i.p. injected 30 minutes later with either 5.0 mg/kg SB-216763 or vehicle (n = 10/group). Rats continued to receive daily injections for the first 5 days after injury during which time they were tested for their vestibulomotor and motor performances (Figure 7A). Drug administration was continued for 5 days since previous studies have reported that apoptosis continues for days after TBI [29]. While SB-216763 injected animals did not perform any differently on the balance beam task compared to vehicle-injected control animals (repeated measures two-way ANOVA: F(1,18) = 0.039, p = 0.846 (Figure 7B), they did show significantly less ipsilateral (Figure 7C; repeated measures two-way ANOVA: F(1,18) = 4.74, p = 0.040) and contra- lateral (Figure 3D; repeated measures two-way ANOVA: F(1,18) = 9.97, p = 0.004) foot faults as compared to their vehicle- injected counterparts. SB-216763 treatment did not have any significant influence on post-injury weight loss (Figure 7E; Figure 5. Post-injury administration of lithium reduces hippo- campal neuronal cell loss. A) Representative photomicrographs of NeuN immunoreactivity from the dorsal, ipsilateral hippocampi of a vehicle- and a lithium-treated animal. Scale bar = 500 mm. DG: dentate gyrus. The outlined area indicates the field in which the cell counts were carried out. B) High-magnification image showing NeuN immunoreac- tivity within the CA3 subfield from a vehicle- and a lithium-injected, injured animal. Scale bar = 200 mm. Lithium treatment C) significantly reduced CA3 neuronal loss, but not D) cortical contusion volume after TBI. *, p#0.05 by Student’s t-test. Veh: vehicle; Li: lithium. Data are presented as the mean 6 SEM. doi:10.1371/journal.pone.0024648.g005 repeated measures two-way ANOVA: F(1,18) = 0.282, p = 0.602). All animals had normal vestibulomotor and motor function by day 7 post-injury (data not shown). When evaluated for their performance in the standard hidden platform version of the Morris water maze task (beginning 14 days post-injury), no difference in learning was detected between injured rats treated with 5.0 mg/kg SB-216763 and those treated with vehicle (Figure 8A; repeated measures two-way ANOVA: F(1,18) = 1.639, p = 0.217). Similarly, when a probe trial was given 24 hr after the completion of training, no differences were seen in the latency to cross (vehicle: 36.6867.24 sec; SB-216763: 42.8166.88 sec, p = 0.725) or the number of platform crossings (vehicle: 1.3060.40 crosses; SB-216763: 0.9060.28 crosses, p = 0.50) between the two groups (Figure 8B). Figure 8C shows that while both the vehicle (one-way repeated measures ANOVA: F(3,27) = 3.186, p = 0.040) and SB-216763 (one-way repeated measures ANOVA: F(3,27) = 4.019, p = 0.017) treated groups displayed significant preference for the target quadrant (I) relative to the starting quadrant (III), there was no significant difference between the two groups in their quadrant preferences (two-way repeated measures ANOVA: F(1,18) = 0.971, p = 0.338). Interest- ingly, when a short term memory test was given at 30 minutes following training, SB-216763 injected animals crossed the platform location significantly more than the vehicle-injected controls (vehicle: 1.1060.35 crosses;SB-216763: 2.3060.42 cross- es, p = 0.042; Figure 8D). No difference in latency (Figure 8D) nor quadrant preference (data not shown) was observed between the two groups during the short-term memory probe trial. Consistent with the lack of improvement in the water maze, histological PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24648