Figure 1.
Time course of MMP gelatinolytic activity after TBI in mice.
A. Representative zymograms of mouse cortical tissue at different time points post-trauma. Gelatin zymography showed gelatinolytic bands representing the latent (proMMP-9) and activated form (act.MMP-9) of MMP-9 in different time courses. B and C. Densitometry analysis of gelatinolytic bands shown in (A) representing the proMMP-9 (B) and act.MMP-9 (C). n = 5 at each time point; *, **, ***, and ****, p<0.05, 0.01, 0.001, and 0.0001, respectively, by one-way ANOVA using Dunnett’s multiple comparison test; data are expressed as means ± SEM.
Figure 2.
Attenuation of MMP-9 gelatinolytic activity after SB-3CT treatment.
A. Representative zymograms comparing cortical MMP-9 levels in SB-3CT versus vehicle-treated mice at 7 days post-trauma. Gelatin zymography revealed increased levels of proMMP-9 and act.MMP-9 in the lesioned cortex of vehicle-treated mice, whereas in SB-3CT-treated mice that activity was significantly attenuated. B–C. Densitometry measurements of proMMP-9 (B) and act.MMP-9 (C) at 7 days post-trauma. n = 7 in vehicle-treated, and 6 in SB-3CT-treated mice; * and ***, p<0.05 and 0.001, respectively, comparing lesioned to contralateral cortex in vehicle-treated mice; # and ##, p<0.05 and 0.01, respectively, comparing the difference between the contralateral and lesioned cortex after SB-3CT treatment to that in vehicle-treated mice by one-tailed, unpaired Student’s t-test; data are expressed as means ± SEM.
Figure 3.
Brain and plasma concentrations versus time curves of SB-3CT and p-OH SB-3CT after repeated 25 mg/kg i.p. administration of SB-3CT to mice.
Samples were analyzed by reversed-phase UPLC/ESI with MRM. SB-3CT and its active metabolite p-OH SB-3CT were rapidly absorbed and distributed to the brain. Brain levels of SB-3CT were above its Ki for MMP-9, while those of p-OH SB-3CT were below its Ki for MMP-9, suggesting that the efficacy seen in the TBI model is probably due to the ability of the parent SB-3CT to cross the BBB.
Table 1.
Concentrations and pharmacokinetic parameters of SB-3CT and p-OH SB-3CT after repeated i.p. administration of SB-3CT to mice.
Table 2.
Regional brain distribution of SB-3CT and p-OH SB-3CT after repeated i.p. administration of SB-3CTb.
Figure 4.
Histopathological quantification of lesion volumes in cresyl violet-stained brain sections at 7 days post-trauma.
A. Representative cresyl violet-stained coronal brain sections from vehicle and SB-3CT-treated mice marked with their coordinates to Bregma. The black area in each section shows the contralateral hemisphere superimposed on top of the lesioned hemisphere to visualize the brain damage regions. B. Stereological scatter-plot of lesion areas in the cresyl-violet stained sections of vehicle and SB-3CT-treated mice at 7 days post-trauma. Each data point represents the lesion area in one cresyl violet-stained brain section, and plotted according to the rostro-caudal axis of the brain coordinate to Bregma. A second degree polynomial was generated to fit data points to visualize data trends. The graphs indicate a difference in lesion area between vehicle and SB-3CT-treated mice. C. Quantification of cortical lesion volume at 7 days post-trauma in the SB-3CT-treated mice compared to the vehicle-treated mice. n = 6 in each group; *, p<0.05 by one-tailed, unpaired Student’s t-test. Data expressed as mean ± SEM.
Figure 5.
SB-3CT treatment protects cortical and hippocampal neurons from dendritic degeneration at 7 days after TBI.
A. Representative photomicrographs of mouse cortical region in sections stained with either cresyl violet (top row), neuronal markers NeuN and MAP-2 (second row) and merged images with Hoechst dye counterstaining (bottom row) showing neuronal cell death and dendritic degeneration. SB-3CT treatment resulted in less irregular, darker stained neurons compared to the vehicle-treated mice (top row). In addition, more neurons with well-defined dendritic processes (white arrows) were seen in the contralateral cortex compared to the lesioned ones (second row). Scale bar (both black and white) = 50 µm. B. Quantification of neuronal cells with dendrites. Numbers of neurons with dendrites were counted from a total of approximately 800–1000 cells in each hemisphere. A marked difference in the percentage of neurons with dendrites was seen between contralateral and lesioned cortex. The percentage of neurons with dendrites was significantly higher in the lesioned cortex of SB-3CT-treated mice compared to that of vehicle-treated mice; n = 5 for each group; ***, p<0.001, comparing the lesioned to contralateral cortex; #, p<0.05, comparing the difference between the contralateral and lesioned cortex after SB-3CT treatment to that in vehicle-treated animals using a one-tailed, unpaired Student’s t-test. Data are expressed as mean ± SEM. C. Comparison of dendritic degeneration in the lesioned and contralateral CA3 subregion of the hippocampus. Neuronal cells in the lesioned CA3 appear in condensed, irregular shape (white arrowheads), while cell bodies in the contralateral region as well as after SB-3CT-treatment appear intact in round shape with dendritic processes (white arrows), indicating that SB-3CT protects against dendritic degeneration from traumatic insult. Scale bar = 50 µm.
Figure 6.
SB-3CT suppressed microglia activation and astrogliosis 7 days post-trauma.
A. A representative microphotograph showing the cortical area for glial cell activation analysis. B. photomicrographs of cortical region in coronal sections of mouse brain immunofluorescently stained with microglial marker CD11b and astrocytic marker GFAP showing increased astroglial activity 7 days post-trauma. SB-3CT treatment attenuated activation of microglia and astrocytes in the lesioned cortex. Scale bar = 50 µm. C. Numbers of the CD11b positive microglia were counted from a total of approximately 1600 cells in the area showed in A. The number of CD11b positive microglia was significantly lower in the lesioned cortex of SB-3CT-treated mice compared to vehicle-treated animals; n = 3 for each group; *, p<0.05 by one-tailed, unpaired Student’s t-test. Data are expressed as mean ± SEM.
Figure 7.
Protective effect of SB-3CT against long-term cortical damage and motor deficits after TBI.
A. Histopathological quantification of cortical lesion area in cresyl-violet stained brain sections from vehicle and SB-3CT-treated mice at 30 days post-trauma. Cortical lesion areas were markedly smaller in SB-3CT-treated mice compared to the vehicle-treated mice. B. Quantification of cortical lesion volume in vehicle and SB-3CT-treated mice at 30 days post-trauma. **, p<0.01 by one-tailed, unpaired Student’s t-test; n = 7 in vehicle-treated, 8 in SB-3CT-treated mice. Data are expressed as mean ± SEM. C. Beam-walking test. SB-3CT-treated mice committed significantly fewer foot faults compared to vehicle-treated animals on days 7 and 14; *, p<0.05, **, p<0.01; but no significant difference between SB-3CT-treated and sham groups, by two-way repeated-measures ANOVA, Bonferroni post tests; n = 11 in sham, 10 in vehicle-treated, and 11 in SB-3CT-treated mice. Data are expressed as mean ± SEM. D. Correlation between cortical damage and motor deficits. Cortical damage areas between 7 days and 30 days after TBI were not significantly different. Cortical lesion area at 30 days and beam-walking foot faults at 7 days were correlated by one-tailed Pearson correlation test, Pearson r = 0.6192, p<0.01; n = 6 in sham, 6 in vehicle-treated, and 8 in SB-3CT-treated mice, showing that the correlation is significant. These three groups–sham, vehicle-treated and SB-3CT-treated mice, display good separation.
Figure 8.
Protective effect of SB-3CT against long-term hippocampal damage and cognitive deficits after TBI.
A. Quantification of hippocampal lesion volume in vehicle and SB-3CT-treated mice, 30 days post-trauma. **, p<0.01 by one-tailed, unpaired Student’s t-test; n = 7 in vehicle-treated, 8 in SB-3CT-treated mice. Data are expressed as means ± SEM. B. Barnes maze acquisition. Testing consisted of 10 trials (2 trials/day) over 5 days. Latency (sec) of each trial was measured. Two-way repeated-measures ANOVA revealed a significant interaction (p = 0.0149) and significant main effects of days (p<0.0001) and groups (p = 0.0011). SB-3CT treated mice performed significantly better than vehicle-treated mice on days 22 and 23 after TBI; *, p<0.05, **, p<0.01; n = 11 in sham, 10 in vehicle-treated, and 11 in SB-3CT-treated mice. Data are expressed as means ± SEM. C. For analysis of memory acquisition in the maze, the latency AUC over 10 trials was calculated for each animal, and group comparisons were performed by one-way ANOVA, Dunnett’s post test, showing that SB-3CT ameliorates cognitive deficits after TBI; *, p<0.05 vs. vehicle-treated; n = 11 in sham, 10 in vehicle-treated, and 11 in SB-3CT-treated mice. Data are expressed as means ± SEM. D. Correlation between hippocampal damage and memory deficits. Hippocampal lesion volumes in 30 days and AUC were correlated by one-tailed Pearson correlation test. Pearson r = 0.5817, p<0.01; n = 6 in sham, 6 in vehicle-treated, and 8 in SB-3CT-treated mice. These data indicated significant correlation and three groups: sham, vehicle-treated, and SB-3CT treated showed good separation.