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using the blocking Kit Avidin/Biotin (Vector) and sections were incubated with Lycopersicon esulentum tomato lectin (Sigma) which stains microglia and macrophages. The immu- noreactions were visualized with LSAB-2 System-HRP (Dako- Cytomation, Denmark) and DAB (Vector) was used as chro- mogen. Sections were finally counterstained with hematoxylin. For all captured images, each square subdivi- sion was counted if it contained at least one labeled element, defined as a labeled cell body and/or its processes. The tissue section, which exhibited the greatest number of either BDNF or synaptophysin positive structures, was selected for statisti- cal analysis (39). The scores of the areas studied represented subjective assessments of fluorescence intensity and numbers of labeled cells, as follows: 0: no labeling observed in any square subdivision; 1 (): 1–10 square subdivisions positive; 2 (): 11–20 square subdivisions positive; 3 (): 20 square subdivisions positive. The scores either for astrogliosis or microglia represented subjective assessments of staining intensity and numbers of labeled cells, as follows: 0: no labeling observed; 1 (): small number of cells that are only weakly labeled; 2 (): moderate number of cells that are clearly labeled; 3 (): large number of intensely labeled cells present (40). Two independent observers performed all quantitative assessments. In cases where significant discrepan- cies were seen between the two observers, the evaluation was repeated by a third one. Sections were examined under either light or fluorescent microscope (Zeiss Axioplan 2, Thorn- wood, NY, USA).

For Western blot analysis, 16 d post CHI, mice were decapitated, the brains removed, and the hippocampus rap- idly dissected and frozen in liquid nitrogen. The tissues were homogenized in homogenization buffer containing 320 mM sucrose, 1% SDS, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA and protease inhibitor cocktail (Sigma). Homog- enates were centrifuged at 1000g for 5 min to remove nuclei and large debris and the supernatant and the amounts of protein determined by BCA (Pierce). Equal amounts of protein (50 g/lane) were separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were probed with anti-BDNF (Chemicon, Temecula, CA, USA), and anti-tubulin (Sigma). Immunoreactivity was de- tected with enhanced chemiluminescence (Pierce, Rockford, IL, USA) and processed using the NIH Image software.

severity of injury was comparable between the vehicle- and DCS-treated mice. Mice with similar initial NSS (mean sem 6.780.40, and 6.990.39; n9/group) were then assigned to receive vehicle (saline) or DCS, respectively. Prior to treatment initiation, 24 h after CHI, NSS of both groups was also similar, as expected (6.560.29 and 6.780.36, respectively). Neurological recovery was followed for 3 more weeks, and the trend toward better recovery of the DCS-treated group, seen at day 8, became highly significant at day 15 and 22 (15 d P 0.05; 22 d P 0.02 vs. vehicle treated mice; Fig. 1A).

DCS facilitates recovery of cognitive function

DCS treated mice also performed significantly better than vehicle treated controls in the object recognition test from day 9 and at 16 and 23 d after CHI, at a dose which had no effect on performance in naive animals. Both groups of mice spent a similar proportion (50%) of time exploring two identical objects in an observation cage at baseline. Four hours after baseline exposure to the identical objects, the mice were rein- troduced into the cage in which one of the two “old” objects was replaced by a novel object, similar in size, color and material to the original one. The vehicle- treated CHI mice spent a similar proportion of their time with the old and new object (Fig. 1B), with no significant preference for the novel object, while the DCS-treated CHI mice increased their exploration of the novel, compared to the familiar object, such that at day 3 a trend (P0.1) toward longer exploration time was already noted. From day 9 and on, the DCS-treated mice spent the same proportion of the exploration time at the novel object (75%) as intact untreated mice, significantly longer than the vehicle-treated ani- mals (Fig. 1B; P0.02 vs. vehicle-treated mice).

Statistical analysis

Impaired LTP following CHI was recovered in DCS- injected mice

Data are expressed as mean sem. A commercial statistics package (Graph Pad Prism version 3.03) was used for deter- mining statistical significance. Significance was determined using the nonparametric Mann-Whitney test for NSS values and one way analysis of variance (ANOVA) followed by student’s t tests for ORT results. Paired pulse ratio was compared using Student’s t test. Histological and immuno- histochemical data were semi quantitative and statistics were used for original data sets. Thus, where appropriate, the Pearson’s 2 or Fisher’s exact test were applied as mentioned in the text. Analysis of the differences in BDNF levels (den- sitometric scans normalized to tubulin) from Western blot was performed by Student’s t test.


DCS facilitates recovery of motor function

One hour after CHI the NSS was evaluated (see Fig. 1A) and mice were randomized into two groups such that

Previous studies have shown that TBI results in alter- ation of synaptic plasticity in the hippocampus that may be responsible for TBI-induced deficits in learning and memory (15, 16, 18, 41–43). If the impairment in the object recognition test following CHI and the improved ability of CHI mice to perform this task after DCS injection (Fig. 1B) are due to changes in synaptic plasticity, it is likely that hippocampal long term poten- tiation (LTP) after CHI will be impaired and that injection of DCS will recover the impaired LTP. Field excitatory post synaptic potentials (fEPSPs) were re- corded in the CA1 region of the hippocampus from slices that were prepared from animals 16 d post DCS injection, when maximal recovery in the object recog- nition test was observed (Fig. 1B). Recordings were made in sham mice, CHI mice that were injected with vehicle (CHIVehicle) and CHI mice that were in- jected with DCS (CHIDCS). When a stable base line was established (Fig. 2A, traces 1, 3 and 5), LTP was induced by tetanic stimulation of afferent fibers (Fig.


Vol. 21

July 2007

The FASEB Journal


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