Figure 1.
Cell distribution of p-Akt following controlled cortical impact (CCI) at day 1 post-injury.
Cellular localization of (A) p-Akt Ser473, (B) p-Akt Thr308 in the peri-contusion margin, and (C) p-Akt Ser473 and (D) p-Akt Thr308 in the contralateral hemisphere 1 day post-injury, observed by immunofluorescence labeling. p-Akt immunoreactivity is shown in red, and immunolabeling of NeuN (a cell marker for neurons), F4/80 (a cell marker for microglia), or GFAP (a cell marker for astrocytes) is shown in green. Yellow labeling indicates co-localization. Both p-Akt Ser473 and Thr308 were mainly expressed in neurons, but rarely in astrocytes or microglia. Sections were stained with DAPI (blue) to show all nuclei. The scale bar is 50 µm.
Table 1.
Metabolic characteristics of the sham control, mice treated with saline and salidroside.
Figure 2.
Effects of different doses of salidroside on behavioral outcomes and brain edema in contusion-injured mice.
(A) Both 20 mg/kg salidroside (SALD 20) and 50 mg/kg salidroside (SALD 50) treatments reduced modified neurological severity score (mNSS), and the mNSS scores in the SALD 20 group were significantly lower compared with the corresponding vehicle group at days 4, 14, 21, and 28 (all P<0.05). The SALD 50 group had significantly lower mNSS scores from day 14 (P<0.05 on days 14, 21 and 28). (B) Both SALD 20 and SALD 50 groups had better rotarod performance compared to the vehicle group. The SALD 20 group had significantly better performance over the whole observation period (all P<0. 05), and SALD 50 significantly improved the rotarod outcome at days 14 and 28 (both P<0. 05). (C) There was significant difference between the SALD 20 and the vehicle groups at days 4 and 21 (P<0.05 and 0.01, respectively) for the beam walk latency. The treatment effect was significant only at day 4 for the SALD 50 group (P<0. 05). Values are presented as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated injured mice. (n = 8–10 mice/group at each time point for behavior tests, repeated measures two-way ANOVA). (D) SALD 20 treatment reduced brain water content in the ipsilateral hemisphere compared to the vehicle group at day 1. Values are presented as mean ± SEM; #P<0.05 versus sham controls, and *P<0.05 versus vehicle-treated injured mice (n = 6 mice/group for brain water content, one-way ANOVA).
Figure 3.
Effects of different doses of salidroside on histological outcomes in contusion-injured mice.
(A) Representative cresyl violet-stained brain sections of vehicle-treated, 20 mg/kg (SALD 20) and 50 mg/kg salidroside (SALD 50)-treated mice 28 days post-injury, showing a pronounced loss of tissue in the injured hemisphere. Scale bar is 1 mm. (B) Quantification revealed significantly smaller contusion volumes in SALD 20-treated mice compared with vehicle-treated mice at day 28. (C) Both SALD 20 and SALD 50 caused significantly greater preservation of brain tissue compared with the vehicle group at day 28. (D) A representative Fluoro-Jade B (FJB)-stained brain section of a core contusional region at 0.74 mm from the bregma at day 1. Quantification analysis indicated that SALD 20-treated mice had significantly fewer degenerating neurons than vehicle-treated mice in the cortical contusion margin at day 1 post-injury. The total number of FJB-positive cells is expressed as the mean number per field of view (0.8 mm2). The scale bar is 50 µm. Values are presented as mean ± SEM; *P<0.05, **P<0.01 versus vehicle-treated injured mice (n = 9 mice/group for cresyl-violet staining, one-way ANOVA and 5 mice/group for FJB histochemistry, Student’s t-test).
Figure 4.
Effects of 20 mg·kg−1 salidroside on apoptosis in contusion-injured mice.
(A) Representative terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining (green)- and DAPI (blue)-stained brain sections of a 20 mg/kg salidroside (SALD 20)-treated mouse and a vehicle-treated mouse at day 1 post-injury. (B) Quantification showed that SALD-treated mice had significantly fewer TUNEL-positive cells around the injured cortical areas at day 1 than observed in the vehicle group. The percentage of TUNEL-positive cells is expressed as the number of TUNEL-stained nuclei/the total number of DAPI-stained nuclei. Sections were stained with DAPI (blue) to show all nuclei. The scale bar is 100 µm. Values are presented as means ± SEM; *P<0.01 versus vehicle-treated injured mice (n = 6 mice/group). (C) Representative immunoblots of cleaved caspase-3, cytosolic cytochrome C (CytC) and Smac/DIABLO in the ipsilateral hemisphere from sham-injured, vehicle-treated and SALD 20-treated mice at 1 and 3 days post-injury. Bar graph of densitometric analysis of bands showing a significant decrease of (D) cleaved caspase-3 (E) cytosolic CytC and (F) cytosolic Smac/DIABLO protein levels in the ipsilateral hemisphere of SALD-20 treated mice at 1 and 3 days post-injury, compared with vehicle-treated mice. Values are presented as mean ± SEM; ##P<0.01, ###P<0.001 versus sham controls, and *P<0.05, **P<0.01 versus vehicle-treated injured mice (n = 6 mice/group at each time point, repeated measures two-way ANOVA).
Figure 5.
Effects of 20 mg·kg−1 salidroside on endogenous survival signals in the cortex of contusion-injured mice.
(A, B, C) Representative immunoblots and densitometric analysis showed a significant increase in p-Akt Ser473 level in the ipsilateral cortex of 20 mg/kg salidroside (SALD 20)-treated injured mice at day 1 compared with vehicle-treated injured mice. The level of p-Akt Thr308 was increased following SALD 20 treatment at day 3 compared with vehicle-treated injured mice. (D, E, F) Representative immunoblots and densitometric analysis showed that there were no differences in the levels of p-Bad and p-FOXO1 in the ipsilateral cortex between the SALD 20 and vehicle groups at day 1 or 3. (G, H, I) Representative immunoblots and densitometric analysis showed that the mitochondrial Bcl-2/Bax ratio significantly increased in SALD-20 treated injured mice but no difference was found in the total Bcl-2/Bax ratio. Values are presented as mean ± SEM; #P<0.05, ##P<0.01 versus sham controls, and *P<0.05 versus vehicle-treated injured mice (n = 6 mice/group at each time point, repeated measures two-way ANOVA for p-Akt Ser473, p-Akt Thr308, p-Bad, and p-FOXO1 levels; one-way ANOVA for Bcl-2/Bax ratio).
Figure 6.
Effects of 20 mg/kg salidroside on apoptotic and survival signals in the hippocampus of injured mice.
Representative immunoblots and densitometric analysis showed that (A, B, C, D) levels of cleaved caspase 3, p-Akt Ser473, p-Akt Thr308, (E, F, G) p-Bad and p-FOXO1 in ipsilateral hippocampal samples from injured animals at post-injury day 1 or 3 were not significantly different from those in sham-injured animals (n = 6 mice/group at each time point, repeated measures two-way ANOVA).
Figure 7.
The effects of inhibition of the PI3K/Akt signaling pathway.
(A, B, C, D, E, F) Representative immunoblots and densitometric analysis in the cortex from sham-injured mice or mice subject to cortical impact injury, infused intracerebroventricular (icv) with saline (vehicle) or LY294002. Mice received either icv pretreatment of LY294002 followed by 20 mg/kg salidroside (SALD 20) after injury (S20Y group) or icv pretreatment of saline followed by SALD 20 after CCI (S20V group). LY294002 did not affect Akt, Bad, or FOXO1 phosphorylation in sham-operated mice, but it significantly abolished SALD 20-induced preservation of p-Akt Thr308 and p-FOXO1 levels in the ipsilateral hemisphere at 1 day post-injury. The levels of p-Akt Ser473 and p-Bad were similar between the S20V and S20LY groups. Values are presented as mean ± SEM; *P<0.05, **P<0.01 versus sham + vehicle (ShamV), and &P<0.05 versus sham + LY294002 (ShamLY), and #P<0.05 versus S20V group (n = 6 mice/group, one-way ANOVA). (G) Representative cresyl violet-stained brain sections of S20V and S20LY mice at 28 days post-injury. Scale bar is 1 mm. (H) Quantification analysis showed that there was a significant decrease in the residual cerebral tissue ratio in the S20LY group compared with the S20S group. Values are presented as mean ± SEM; *P<0.05 versus S20V group (n = 6 mice/group, Student’s t-test).
Figure 8.
The effect of different lag times of post-treatment with 20mg/kg salidroside.
(A, B) Mice treated with 20 mg/kg salidroside (SALD 20) had significantly fewer degenerating neurons than vehicle-treated mice in the cortical contusion margin at 1 day when administered at both 3 and 6 h post-injury. The total number of Fluoro-Jade B (FJB)-positive cells is expressed as the mean number per field of view (0.8 mm2). (C, D, E, F) Representative immunoblots and densitometric analysis showed that SALD 20 treatment significantly decreased cleaved caspase-3 level and increased p-Akt Ser473 level but no significant difference was found in p-Akt Thr 308 level at day 1 when administered at 3 h post-injury. Values are presented as mean ± SEM; #P<0.05, ##P<0.01, ###P<0.001 versus sham controls, and *P<0.05, ***P<0.001 versus vehicle-treated injured mice (n = 6 mice/group, Student’s t-test for FJB staining; one-way ANOVA for immunoblots).
Figure 9.
Protective effects of salidroside on stretch-induced cytotoxicity in primary cultured neurons.
Treatment with salidroside at different concentrations (0.1, 1, 10, 20, and 50 µM) ameliorated stretch-induced cell viability loss in a dose-dependent manner. Values are presented as mean ± SEM of 3 independent experiments (n = 3); ***P<0.001 versus control, ###P<0.001 versus stretch injury alone. All data were representative of 4 independent experiments.
Table 2.
Antibodies used in immunofluorescence and western blot.