recommended by the vendor (Vector Laboratories, Burlingame, CA). Following extensive washing, immunoreactivity was detected using a chemiluminescence system and quantified using Image J (freely available through NIH). Assessment of motor function All behavioral tests were conducted by an experimenter blind to the treatment groups. A vestibulomotor (beam balance) and a motor skill task (paw placement) were used to determine animals’ motor performance on days 1–4 post-injury. For beam balance, rats were pre-assessed by placing them on a narrow wooden beam (1.5 cm wide) and measuring the duration they remained on the beam for up to 60 seconds. Animals were given repeated training until capable of balancing on the beam for the entire 60 sec period for 3 consecutive trials. Following injury, animals were given three daily trials during which the length of time spent on the beam was recorded. Paw placement was evaluated by placing the animal on a wire grid (opening size of 262 cm) and counting the number of foot faults out of a total of 50 steps. A foot fault was defined as when a front paw misses and appears below the plane of the grid. Paw placement was repeated three times to give an average daily score. Assessment of cognitive function Rats were tested for their cognitive performance using the standard hidden platform version of the Morris water maze [17,19–21]. All animals had recovered from the TBI-associated motor dysfunction prior to performing the cognitive testing. Animals were given 4 consecutive training trials per day with an inter-trial interval (iti) of 4min. If the animal failed to locate the platform within 60 sec on any given trial, it was led there by the experimenter. Thirty minutes after the last daily training trial, animals were given a probe trial to measure quadrant preference and platform localization. Twenty-four hours following the last day of training, animals were again tested in a probe trial to measure quadrant preference and platform localization. Move- ment within the maze was monitored using a video camera linked to tracking software (Ethovision, Noldus Information Technology, Leesbury, VA, USA). Contusion volume measurement Following the completion of the behavioral studies, approxi- mately 28 days following the injury, animals (n = 10/group) were deeply anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed, post- fixed overnight in perfusant, then cryoprotected in a 30% sucrose solution. Cortical tissue loss was estimated essentially as described previously [22], by experimenters kept blind with respect to the treatment groups. In brief, cryosections (40 mm thickness) spanning the rostral-caudal extent of the injured cortex were selected and stained with cresyl violet by an experimenter given only the animal’s identifier code. Images of the resultant slides were then used for tissue loss measurement by a second experimenter. The area of cortical tissue loss for each section was carefully outlined using Adobe Illustrator, with the area of the resultant outlines quantified by Image J, from the National Institutes of Health. Contusion volume was calculated using the equation A1(0.5X1)+A2(0.5X1 +0.5X2)+An-1(0.5Xn-1 +0.5Xn)+ An(0.5Xn) where A is the area (mm2) of the contusion for each slice, and X is the distance (mm) between two sequential slices. Once the contusion volume had been calculated for each animal, the blind code was broken and group differences assessed. Glycogen Synthase Kinase-3 and TBI Immunohistochemistry For post-TBI histological evaluation, tissue sections generated for the measurement of cortical contusion volume were used for immunohistochemistry. Free-floating slices were incubated over- night in primary antibody (0.5–1.0 mg/ml) in TBS containing 2% BSA and 2.5% normal goat serum. After extensive washing, immunoreactivity was detected using species-specific secondary antibodies coupled to Alexafluors. These fluorescently-labeled tissue sections were evaluated by a blind observer to identify any potential differences between the treatment groups. As visible neuroprotection was detected following lithium administration, stereological cell counts were carried out. For cell counts, NeuN immunostaining was performed using a colorimetric detection system consisting of a secondary antibody conjugated to horseradish-peroxidase and diaminobenzadine (DAB) as the chromagen. This was done to avoid the photobleaching often associated with high magnification imaging of fluorescent immunohistochemistry. Stereology A blind counting methodology was employed for determination of NeuN positive cells in the CA3 layer of the ipsilateral hippocampus in lithium chloride- and vehicle-treated animals (n = 5/group). Every tenth section through the ipsilateral dorsal hippocampus was processed for NeuN immunoreactivity using colorimetric detection as described above. NeuN positive cells within the CA3 subfield were counted using the optical dissector technique using Stereo Investigator (MicroBrightField Bioscience, Williston, VT) [23]. The CA3 subfield was differentiated from the CA2 by the density of cells within these layers. As there is no morphological distinction between the CA3 and CA4 subfields using NeuN staining, these subfields were divided by dropping a vertical line from the lateral edge of the inner blade of the dentate gyrus. Cells in the outermost planes of focus (5 mm) were omitted to avoid counting cell caps. The number of NeuN-labeled cells in approximately twenty, computer chosen areas within the CA3 cell layer was scored for each section. The counting frame was 1086108 mm. The size of the counting frame, and the number of grid sections was determined based on preliminary cell counts. The number of NeuN labeled cells/mm3 for each section was obtained from the estimated cells divided by the contour volume. The number of cells/mm3 for each animal was calculated as the average of the number of cells/mm3 from each section examined. Statistical Analysis For evaluation of behavioral data, repeated measures analyses of variance (two-way or one-way as appropriate) and t-tests were utilized to determine statistical differences. A Holm-Sidak method for multiple comparisons post-hoc test was used to determine data points with significant differences. Dose response western data were evaluated using a one-way ANOVA followed by a Dunnet’s post-hoc test. For data that did not pass a Shapiro-Wilk normality test, appropriate non-parametric analysis was performed. Data were considered significant at p#0.05 and presented as Mean 6 Standard Error of the Mean (S.E.M.). Results TBI causes transient inhibition of the GSK-3 pathway To determine the influence of TBI on GSK-3 activity, western blots were performed to examine the phosphorylation of LRP6, GSK-3ß, and ß-Catenin in hippocampal extracts prepared at various time points following injury (n = 5/time point). Figure 1D shows images of representative westerns illustrating the phosphor- PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24648