Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside_significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside_also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro.

We then assessed the preservation of brain tissue in CCI mice following the administration of LY294002. The histological analysis showed that there was a significant decrease in the residual cerebral tissue ratio (71.0±6.3%) in the S20LY group compared to the S20S group (80.5±5.8%; P<0. 05; Fig. 7 G and H) at day 28 post-injury.

Mice were anesthetized and positioned in a stereotaxic frame. LY294002 (200 nM in 0.5 µL of saline) or equal amount of physiological saline was intracerebroventricularly (icv) injected at 30 min before CCI as previously described [56]. A 1 mm cranial burr hole was drilled into the skull. A 30-gauge needle on a Hamilton syringe was implanted into the lateral ventricle using stereotactic coordinates: 1.5 mm posterior to bregma and 1.0 mm right lateral to the midline, 2 mm in depth. LY294002 or saline was infused into the brain at a rate of 0.05 µL/min over 10 min with an infusion pump (KDS 310, KD Scientific Inc., Holliston, MA, USA), and the needle was left in place for an additional 20 min to prevent reflux. After the needle was removed, CCI procedure was conducted immediately. Sham-operated mice were infused icv with saline, but were not subjected to CCI.

Three sets of experiments were performed. The first set was to investigate the protective effects of salidroside. All animals were randomized into 1 of 3 groups (sham injury, CCI + vehicle, CCI + SALD 20). Surgery/injury, behavioral testing, and tissue analyses were performed by different individuals. Behavioral testing and tissue analyses were performed by individuals blinded to the treatment group. Salidroside (Cayman Chemical, Ann Arbor, MI, USA) dissolved in normal saline (0.25 mL) or a corresponding volume of vehicle (saline) was administered intraperitoneally 10 min following injury. Testing after injury was completed as follows: 1) behavioral testing at days 1, 4, 7, 14, 21, and 28 (n=8–10 mice/group); 2) cresyl violet staining at day 28 (n=9 mice/group); 3) histology, determination of brain water content, and western blot analysis at day 1 or 3 (n=5–6 mice/group). To determine the optimal dose of salidroside, a pilot study was performed using 2 different doses (20 and 50 mg/kg) administered 10 min after injury, and neurological deficits and tissue damage were evaluated as the main outcomes. The results showed a significant effect of salidroside at 20 mg/kg, with no further increase in efficacy observed at 50 mg/kg. Therefore, on the basis of these data, a dose of 20 mg/kg was chosen for all subsequent experiments. The second set of experiment was to assess the effect of the inhibition of PI3K/Akt with the specific PI3K inhibitor, LY294002 (Cell Signaling Technology, Danvers, MA, USA), on salidroside effects. Animals were randomized into 4 groups. Mice received either: 1) icv pretreatment of LY294002 followed by SALD 20 after CCI (S20Y group); or 2) icv pretreatment of saline (vehicle control) followed by SALD 20 after CCI (S20V group). 3) Mice were infused icv with saline and were subjected to sham injury. 4) Mice were infused icv with LY294002 and were subjected to sham injury. Testing after injury was completed as follows: 1) cresyl violet staining at day 28 (n=6 mice/group); 2) western blot analysis at day 1 (n=6 mice/group). The third set of experiment was performed to examine the neuroprotective time window afforded by salidroside. Mice received intraperitoneal SALD 20 or vehicle (saline) at 3 or 6 h following CCI and were sacrificed 1 day later for FJB staining and western blot analysis (n=6 mice/group).

Following terminal anesthesia, venous blood was collected via direct right atrial puncture. The obtained blood was centrifuged (3500 rpm for 5 min), and the serum was stored at −20°C until analysis. A chemistry analyzer (Synchron Clinical System LX20; Beckman Coulter, Fullerton, CA, USA) was used to measure serum BUN and CRE to assess renal function, and ALT to assess liver function.

Behavioral testing was performed before CCI and at 1, 4, 7, 14, 21, and 14 days after CCI. The battery of tests consisted of the mNSS, rotarod motor test, and beam walk test. Animals were pretrained for 3 days for the rotarod, and beam walk tests.

The mNSS is a composite test of motor, sensory, balance and reflex [57]. One point was recorded for the inability to perform the test or for the lack of a subjected reflex; thus, the higher the score, the more severe the injury. Neurological function was graded on a scale of 0–18 (normal score, 0; maximal deficit score, 18).

An accelerating rotarod was used to measure motor function and balance [58]. Animals were placed on a 3 cm rotarod cylinder, and the time for which the animals remained on the rotarod was measured. Speed was increased from 6 to 42 rpm every min for a duration of 7 min. A trial ended if the animal fell off the rungs or gripped the cylinder and spun around for 2 consecutive revolutions without attempting to walk on the rungs. One hour before CCI, the mean duration of each animal on the device was recorded with 3 rotarod measurements for the preinjury baseline values.

The beam walk test was utilized to evaluate fine motor coordination and function [59]. Mice tended to escape a bright open area by walking along a narrowed wooden beam (0.8×100.0 cm) to enter a darkened goal box at the opposite end of the beam. The latency of the animal to reach the goal box (not to exceed 60 s) was recorded. Three trials were recorded 1 h before CCI as baseline values.

Brain edema was examined by measuring brain water content using the wet−dry/wet brain weight method. Mice were re-anesthetized and decapitated at day 1, a time point associated with maximal edema formation following experimental TBI [22]. Brain water content was measured in a 4mm coronal tissue section of the ipsilateral hemisphere, 2 mm from the frontal pole. Brain samples were immediately weighed on an electric analytical balance to obtain the wet weight and then dried at 100°C for 24 h to obtain the dry weight. The following formula was used for calculation of edema: (wet weight - dry weight)/wet weight ×100%.

After anesthesia, mice were sacrificed by transcardial perfusion with phosphate-buffered saline (PBS) and then their tissues were fixed with 4% paraformaldehyde at day 1 (for FJB staining, TUNEL, immunofluorescent or cresyl violet histology) and day 28 (for cresyl violet histology) post-injury. Brains were collected and post-fixed in 4% paraformaldehyde overnight and transferred to PBS containing 30% sucrose for cryoprotection. Sections were sliced in a cryostat in 10 µm sections from the level of the olfactory bulbs to the visual cortex.

Contusion volumes and residual cerebral tissue ratios were quantified using coronal sections stained with cresyl violet at 20 rostral-caudal levels that were spaced 200 µm apart. Sections were digitized and analyzed using a 1.5× objective and Image J software (Image J, National Institutes of Health, Bethesda, MD, USA). The contusion area was calculated using all cresyl violet-stained sections containing contused brain as previously described [60], and the volume measurement was computed by summation of the areas multiplied by the interslice distance (200 µm). The preservation of cerebral tissue was further evaluated by the ratio of the ipsilateral remaining cerebral hemisphere volume to the volume of corresponding contralateral cerebral hemisphere.

FJB is a polyanionic fluorescein derivative that sensitively and specifically binds to degenerating neurons. Staining was carried out as previously described [24]. Briefly, sections were initially incubated in a solution of 1% NaOH in 80% ethanol for 5 min and then hydrated in graded ethanol (50%, 75%, and 100%; 5 min respectively) and distilled water. Sections were incubated in a solution of 0.06% potassium permanganate for 15 min, rinsed in distilled water for 2 min, and incubated in a 0.001% solution of FJB (Chemicon, Temecula, CA, USA) for 30 min. Sections were observed and photographed under a fluorescence microscope (Olympus BX-51; Olympus, Tokyo, Japan) with blue (450–490 nm) excitation light.

TUNEL assay was operated using a commercial kit that labels DNA fragmentation with fluorescein isothiocyanate (In situ Cell Death Detection Kit; Roche Molecular Biochemicals, Mannheim, Germany). Sections were first incubated in 25 µg/mL proteinase-K in 10 mmol/L Tris-HCl at 37°C for 15 min. After being washed in distilled water and PBS, sections were incubated in 0.3% hydrogen peroxide solution. Each section was incubated with 70 µL of TUNEL reaction mixture containing terminal deoxynucleotidyl transferase for 60 min at 37°C under humidified conditions. Sections were observed and photographed under a fluorescence microscope (Olympus BX-51) with blue (450–490 nm) excitation light. Negative controls were obtained by omission of terminal deoxynucleotidyl transferase.

FJB and TUNEL staining was quantified on stained sections from the injury core at the level 0.74 mm from the bregma. Three sections per animal were photographed by a microscope. FJB- and TUNEL-positive cells were counted by sampling an area of 920×860 µm² in 3 randomly selected, non-overlapping fields with the magnification of 200. The total number of FJB-positive cells was expressed as the mean number per field of view. Quantification of TUNEL staining was expressed as the percentage of nuclei that were stained by the TUNEL assay divided by the total number of DAPI–stained nuclei.

Brains of injured or sham animals were collected 1 day and 3 days post CCI or sham surgery, and a 4 mm coronal section from the injured area over the parietal cortex and ipsilateral hippocampus were collected. The protein samples were obtained from tissue homogenates in 300 µL of ice-cold reagent (T-PER reagent; Pierce Biotechnology, Rockford, IL, USA) with a complete mini protease inhibitor cocktail (Roche Molecular Biochemicals) centrifuged at 14000g for 30 min. Protein concentration was determined by Bradford reagent spectrophotometrically at A595. Equal amounts of protein samples were denatured in gel-loading buffer at 100°C for 5 min, and then loaded onto sodium dodecyl sulfate-polyacrylamide gels to be separated. Separated proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA), blocked with the use of 5% milk in PBST and probed with primary antibodies. Afterward, the membranes were washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 11000) at 4°C for 1 h. The relative intensity of protein signals was normalized to the corresponding β-actin intensity and was quantified by densitometric analysis with the use of Image J software.

Dissected cortices (prepared as in western blot analysis) were immediately homogenized in 300 µL of ice-cold cytosol extraction buffer (Cytosol/Mitochondria Fractionation kit; Merck, Rockland, Massachusetts, USA) with a protease inhibitor cocktail and DTT. The homogenates were then centrifuged at 700 g for 10 min at 4°C, and the supernatant was further centrifuged at 10000 g for 30 min at 4°C. The 10000 g supernatant was collected as the cytosolic fraction, and the pellet contained the mitochondria. The mitochondrial fraction was resuspended in 50 µL mitochondrial extraction buffer mix containing protease inhibitors and DTT for 10 s and saved as mitochondrial fraction, or maintained intact in PBS at –80°C until use.