Male imprinting control region (ICR) mice (Experiment Animal Centre of Nanjing Medical University, Jiangsu, China) aged between 6–8 weeks, weighing 28–32 g were used in the present study. The experimental procedures were approved by the Animal Care and Use Committee of Nanjing Jinling Hospital and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (Bethesda, MD, USA). The mice were housed on a 12 h light/dark cycle with ad libitum access to food and water.

The model of TBI used in the present study was a weight-drop model (Beyotime Institute of Biotechnology, Shanghai, China), as described by Flierl et al (20). The mice were anesthetized with an intraperitoneal (i.p.) injection of chloral hydrate (1%; 5 ml/kg; Beyotime Institute of Biotechnology) and then placed onto a platform directly below the weight of the weight-drop device. A 1.5 cm midline longitudinal scalp incision was made and the skull was exposed. Subsequent to locating the left anterior frontal area (1.5 mm lateral to the midline on the mid-coronal plane) as the impact area, a 200 g weight was released and dropped onto the skull from a height of 2.5 cm. The mortality rate resulting from apnea was reduced by early respiratory support. The scalp wound was closed using standard suture material, and the mice were returned to cages, where they had ad libitum access to water and food. Sham-injured animals underwent the same procedures, but did not undergo the weight-drop.

The male ICR mice were divided into four groups (n=45 per group): Sham group, TBI group, TBI + dimethyl sulfoxide (DMSO) group and TBI + rapamycin group. In the TBI + rapamycin group, rapamycin (cat. no. S1039; Selleckchem, Munich, Germany) was dissolved in DMSO (50 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and injected into the mice 30 min after TBI (2 mg/kg; i.p.). The mice in the TBI + DMSO group received equal volumes of vehicle (5% DMSO) at 30 min subsequent to TBI.

For isolation of the proteins, the animals were anesthetized with a solution of chloral hydrate (1%, 5 ml/kg) 24 h after TBI and were perfused intracardially with 30–40 ml cold (4°C) heparinized 0.9% saline (Beyotime Institute of Biotechnology). The left, ipsilateral, cerebral cortex (pericontusion) was collected, immediately frozen in liquid nitrogen and then transferred to a −80°C freezer until use. For immunohistochemical analysis, the animals were sacrificed 24 h after TBI in the following way. Following anesthesia, induced with chloral hydrate (1%, 5 ml/kg), the animals were intracardially perfused with 30–40 ml cold heparinized 0.9% saline followed by 20–30 ml cold 4% paraformaldehyde (Beyotime Institute of Biotechnology). The whole brain was removed and immersed in 4% paraformaldehyde overnight at 4°C. For immunofluorescence, the brain was subsequently immersed in 20% sucrose (Beyotime Institute of Biotechnology) followed by 30% sucrose.

The protein concentrations were determined using the Bradford assay (Beyotime Institute of Biotechnology) (21). Equal quantities of protein (50 µg/per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (Beyotime Institute of Biotechnology) electrophoresis and transferred onto polyvinylidene-difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 2 h in blocking buffer (Tris-buffered saline/0.05% Tween 20; TBST; Beyotime Institute of Biotechnology) containing 5% skim milk) and were incubated overnight at 4°C with the following primary antibodies in blocking buffer: Rabbit monoclonal phospho-mTOR (Ser2448) (cat..no. #5536; 1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal phospho-S6 ribosomal protein (Ser235/236) (cat..no. #4858; 1:2,000; Cell Signaling Technology) and rabbit polyclonal GAPDH (cat. no. AP0063; 1:5,000; Bioworld Technology, Minneapolis, MN, USA). Following washing of the membranes three times with TBST (10 min each), the membranes were incubated with polyclonal goat anti-rabbit horseradish peroxidase conjugated immunoglobulin G (cat. no. BS13278; 1:5,000; Bioworld Technology) for 2 h at room temperature. The protein bands were visualized using enhanced chemiluminescence western blotting detection reagents (EMD Millipore) and exposure to X-ray film (Carestream, Xiamen, China). The developed films were digitized using an Epson Perfection 2480 scanner (Seiko Corp., Nagano, Japan). The band density was quantified using Un-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA) and the data were normalized to GAPDH.

For immunofluorescence, serial 8-µm coronal sections were obtained using a cryostat (RM2235; Leica Microsystems GmbH, Wetzlar, Germany). A total of four sets of five evenly spaced (300 µm apart) sections, spanning the injured cortex, were collected from each brain. Based on established immunostaining procedures (22), slides (Beyotime Institute of Biotechnology) were incubated in blocking buffer, containing 10% normal goat serum in phosphate-buffered saline (PBS) and 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 2 h, followed by incubation at 4°C overnight with the primary antibody, rabbit anti-ionized calcium-binding adapter molecule 1 (IBA-1; cat. no. 019-19741; 1:5,000; Wako, Osaka, Japan). On the following day, the slides were washed with PBS three times for 5 min and incubated with the appropriate secondary antibodies for 1 h at room temperature. The slides were then washed three times in PBS. Cover slips were applied using mounting medium. Images of the immunofluorescence were captured using an Axio Observer A1 microscope system (Carl Zeiss, Oberkochen, Germany) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The specificity of the immunofluorescence reaction was evaluated by replacement of the primary antibody with PBS. A total of six visual fields (magnification, ×200) surrounding the contusion in each coronary section were randomly selected, and the mean number of microglia in the six fields was calculated. A total of four sections from each animal were used for quantification. The data for each sample was the mean number of microglia in the four sections. Data are presented as the mean number of microglia per magnification field (magnification, ×200). All analyses were performed by two investigators in a blinded-manner.

The expression levels of interluekin (IL)-1β and TNF-α were analyzed using an ELISA (Biocalvin Company, Suzhou, China), according to the manufacturer's instructions. The protein concentrations were measured using a Bradford assay. Equal quantities of lysate were used for the analyses of TNF-α and IL-1β, with values expressed as pg/mg protein.

Tissue sections were stained with Cresyl Violet (Nissl; Sigma-Aldrich), as described previously (23). Normal neurons have relatively large cell bodies and are rich in cytoplasm, with one or two large round nuclei. By contrast, damaged cells exhibit shrunken cell bodies, condensed nuclei, a dark cytoplasm and numerous empty vesicles (23). The counting of cells was restricted to the lesion boundary zone (where the most damage was observed). A total of six high-power fields (magnification, ×400) in each coronary section were randomly selected, and the mean number of surviving neurons in the six views were calculated for each section using the Axio Observer A1 microscope system. A total of four sections from each animal were used for quantification and the average quantity of the four sections was calculated for each sample. Data are presented as the quantity of neurons per high-power field. All analyses were performed by two investigators in a blinded-manner.

The neurological statuses of the mice were evaluated 24 h and 72 h after TBI using a grip test and neurological severity score (NSS). The grip test was developed to assess the gross vestibulomotor function (24). The mouse was placed on a thin, horizontal metal wire, measuring 45 cm in length, which was suspended 45 cm above a foam pad between two vertical poles. Each mouse was graded on its ability to grip, attach and move, as described in Table I. The grip test was performed in triplicate, with the total score calculated for each mouse. In the NSS, the ability of each mouse to perform 10 different tasks, which demonstrate motor function, balance and alertness, was evaluated. A single point was scored for failing to perform each of the tasks; thus, 0=minimum deficit and 10=maximum deficit (Table II) (3,20). The severity of the injury was defined by the initial NSS, evaluated 1 h after TBI, providing a reliable predictor of the later outcome. All neurobehavioral assessments were performed by two investigators in a blinded-manner.

Each experiment was repeated at least three times and the data are expressed as the mean ± standard error of the mean. For the behavioral assessment, two-way analysis of variance was used followed by a Bonferroni post hoc test. For the other assays, one-way analysis of variance was used followed by Tukey's test. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for the statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

In the grip test, the scores of the rapamycin-treated mice were significantly improved, compared with those of the vehicle-treated mice 24 h and 72 h after TBI (P<0.01 and P<0.05, respectively; Fig. 1A). As shown in Fig. 1B, the NSS scores of the TBI and the TBI + DMSO groups were lower at 72 h than at 24 h. At 24 h following TBI, the NSS scores of the rapamycin-treated mice were significantly lower compared with those of the vehicle treated mice (P<0.01), and at 72 h, the scores of the two groups had improved, maintaining a significant difference (P<0.05).

Nissl staining was used to evaluate neuronal survival in the pericontusional cortex 24 h after TBI (Fig. 2). Compared with the sham group, TBI decreased the number of neurons in the pericontusional cortex (P<0.01). A large proportion of neurons in the TBI group were damaged, exhibiting extensive degenerative changes, which included sparse cellular arrangements, loss of integrity, shrunken cytoplasma and misshapen nuclei. The sham group was observed to contain clear and intact neurons. Rapamycin significantly increased the proportion of surviving neurons at 24 h after TBI (P<0.05) and may, therefore, have alleviated the severity of neuronal degeneration.

To evaluate the association between rapamycin and proinflammatory cytokines, an ELISA was used to detect the protein levels of proinflammatory factors 24 h after TBI (Fig. 3). The expression levels of IL-1β and TNF-α increased significantly following TBI compared with the sham group (P<0.01 and P<0.001, respectively). No difference was observed between the TBI + DMSO group and the TBI group (P>0.05). The expression levels of IL-1β and TNF-α were reduced in the rapamycin-treated groups compared with those in the DMSO-treated group (P<0.05).

The levels of microglial activation were investigated by detecting the immunofluorescence of IBA-1. As shown in Fig. 4, the microglia in the sham group exhibited long branching processes and small cellular bodies. In response to brain injury, the number of microglia increased significantly (P<0.01) compared with the sham group, and the branches of the IBA-1-stained microglia became short, retracted and thick, indicating activated microglia. Compared with the TBI and TBI + DMSO groups, rapamycin injection decreased the number of IBA-1-stained cells (P<0.05) and microglia exhibited smaller cellular bodies.

To confirm the inactivation of the mTOR pathway induced by rapamycin, the protein expression levels of phosphorylated mTOR and S6RP were investigated using western blotting. As shown in Fig. 5, the expression levels of p-mTOR and p-S6RP were significantly increased in the pericontusional cortex 24 h after TBI (P<0.01). No statistically significant differences were identified between the TBI group and the TBI + DMSO group (P>0.05). Compared with the TBI + DMSO group, rapamycin administration markedly decreased the expression levels of p-mTOR and p-S6RP (P<0.05).