All experimental procedures were conducted in accordance with the guidelines of the Chinese council on animal protection (http://www.hebstd.gov.cn/banshi/zxyw/content_74239.htm) and were approved by the Hebei Medical University (Shijiazhuang, China) Committee for the use of animals in research. A total of 150 adult (age, 12–16 weeks) male Sprague-Dawley rats weighing 300–330 g were obtained from Hebei United University Experimental Animal Center (Tangshan, China). The animals were maintained at 21–26°C under a 12-h light/dark cycle, and water and food were provided ad libitum prior to and following surgery or sham surgery. The rat model of TBI was induced using a weight-drop device, as previously described by Marmarou et al (19). Briefly, the rats were anesthetized with 10% chloral hydrate (3 ml/kg). A midline incision was made to expose the skull between the bregma and lambda suture lines and a steel disc (10 mm in diameter and 3 mm thickness) was adhered to the skull using dental acrylic. Then rats were placed on a foam mattress underneath a weight-drop device in which a 350 g weight falls freely through a vertical tube from 1.5 m onto the steel disk. Following injury, the scalp was sutured. Rats were housed in individual cages and placed on heat pads (37°C) for 24 h to maintain normal body temperature during the recovery period. This model is generally associated with 20% mortality within the first 5 min post-injury and no delayed mortality was observed thereafter. The sham-operated animals were anesthetized and had the steel disk attached to them, but they did not receive TBI.

Rats were randomly divided into three groups: Sham operation group (sham, n=30); TBI group (TBI, n=60); and TBI treated with RV group (RV, n=60). RV (Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared by dissolving in 50% ethanol and diluting in physiological saline (2%) at a concentration of 100 mg/kg, and was administered daily via intraperitoneal injection to the rats in the RV group for 5 days beginning immediately following TBI (14). Both the sham and TBI groups received equal volumes of ethanol (2%) by intraperitoneal injection at the same time daily. Each subgroup was composed of five rats and the rats were sacrificed at 1, 3 and 5 days following TBI.

Brain edema was evaluated by analysis of brain water content using the wet-dry weight method, as described previously (20). Briefly, rats were randomly sampled from each group and anesthetized by intra-peritoneal injection with 10% chloral hydrate (3 ml/kg; n=5). The cerebral tissues were then removed and weighed immediately on an electric analytic balance for the wet weight, then dried at 100°C for 24 h to obtain the dry weight. The percentage of water in the tissues was calculated according to the formula: % brain water = [(wet weight - dry weight)/wet weight] × 100.

To evaluate spatial learning and memory, rats were tested in variation of the MWM paradigm (21). The maze consists of a water-filled pool (180 cm diameter, 45 cm high) at 26°C and virtually divided into 4 equivalent quadrants: North (N), west (W), south (S) and east (E). A 2 cm submerged escape platform (diameter 12 cm, made opaque with paint) was placed in the middle of one of the quadrants equidistant from the sidewall and the center of the pool. All experimental rats were trained to find the platform prior to TBI or sham surgery. At the start of a trial, the rat was placed at one of four fixed starting points, randomly facing a wall (designated N, S, E and W) and allowed to swim for 60 sec or until it found the platform. If the animal found the platform, it was allowed to remain on it for 20 sec. If the animal failed to find the platform within 90 sec, it was placed on the platform for 20 sec. The time required (escape latency) to find the hidden platform with a 60 sec limit was recorded by a video camera suspended above the maze and interfaced with a video tracking system (HVS Imaging, Hampton, UK). The test was conducted at 3–5 days following TBI or sham surgery, and each rat was tested for four trials per day for three consecutive days. The average escape latency of the total of the four trials was calculated.

Neurological deficits were evaluated using the Neurologic Severity Score (NSS) on a 10-point scale, as previously described by Chen et al (22) by a researcher blinded to treatment. One point is awarded for failure to perform a particular task, and a score of ten reflects maximal impairment, thus, a normal rat scores zero. Post-injury, the NSS was evaluated at days 1–5.

Western blotting was performed as previously described (23). At scheduled time points, rats were anesthetized with 10% chloral hydrate and decapitated. The brains were quickly removed and the hippocampal tissues were dissected on ice. Total proteins were extracted and the protein concentration was determined using a bicinchoninic acid kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Equal amounts of protein (50 mg) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, Haimen, China). Separated proteins on the gel were transferred onto polyvinylidene fluoride membranes (Roche Diagnostics GmbH, Mannheim, Germany) by a transfer apparatus at 200 mA for 50 min. The membrane was then blocked with 5% fat-free dry milk for 2 h at room temperature. Subsequently, blots were incubated with the following primary antibodies overnight at 4°C: Polyclonal rabbit anti-microtubule-associated protein light chain 3 (LC3) [cat. no. PD014; Molecular & Biological Laboratories Co., Ltd., Nagoya, Japan (dilution, 1:1,000)], polyclonal rabbit anti-Beclin1 [cat. no. JM-3663-100; Molecular & Biological Laboratories Co., Ltd. (dilution, 1:1,000)], polyclonal rabbit anti-p62 [cat. no. ARH4210; Antibody Revolution, Inc. San Diego, CA, USA (dilution, 1:1,000)], monoclonal rabbit anti-postsynaptic density protein 95 (PSD95) [cat. no. ab76108; Abcam, Cambridge, UK (dilution,1:500)], monoclonal rabbit anti-synaptophysin (SYN) [cat. no. ARH4007; Antibody Revolution, Inc. (dilution, 1:500)], polyclonal rabbit anti-β-actin [cat. no. AF7018; Affinity Biologicals, Inc., Cincinnati, OH, USA (dilution, 1:1,000)]. The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG [cat. no. sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA (dilution, 1:5,000)] for 2 h at room temperature, prior to development using an enhanced chemiluminescence detection system [MultiSciences (LIANKE) Biotech Co., Ltd., Hangzhou, China] and the densitometric signals were quantified using an imaging program (Image Lab 4.1; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The immunoreactive bands were normalized to rabbit anti-β-actin polyclonal antibody (dilution, 1:1,000). Results were analyzed using the National Institutes of Health ImageJ 1.41 software (Bethesda, MD, USA).

Rats were perfused transcardially with saline under deep anesthesia [10% chloral hydrate (3 ml/kg) by intraperitoneal injection]. The brain tissues were the fixed using 4% formaldehyde for 24 h, transferred to a 30% sucrose solution [0.1 M phosphate-buffered saline (PBS), pH 7.4] until they sank to the bottom, following which they were embedded in optimal cutting temperature compound. The brains were cut into 15 µm-thick sections coronally from the anterior to posterior hippocampus (bregma −2.0 to −3.5 mm) using a cryostat. Frozen sections were treated with 0.4% Triton X-100 for 30 min, and blocked in normal donkey serum for 1 h. For double labeling, the frozen sections were incubated with a mixture of polyclonal rabbit anti-LC3 (1:100) and monoclonal mouse anti-NeuN [cat. no. MAB377; EMD Millipore, Billerica, MA, USA (dilution, 1:100)] antibodies overnight at 4°C. The following day, the sections were incubated with a mixture of donkey anti-rabbit IgG-fluorescein isothiocyanate (FITC) and donkey anti-mouse IgG-FITC [cat. nos. sc-2090 and sc-2099, respectively; Santa Cruz Biotechnology, Inc. (dilution, 1:1,000)] for 2 h at 37°C in the dark. All cell nuclei were counterstained by 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a laser scanning confocal microscope (Olympus Fluoview™ FV1000; Olympus Corporation, Tokyo Japan). Primary antibodies were replaced with PBS in the negative control group.

Statistical analysis was performed using SPSS software, version 16.0 (SPSS, Chicago, IL, USA). Data are presented as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by the Student-Newman-Keuls post hoc multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

The wet-dry weight method was used to evaluate brain edema. As shown in Fig. 1, TBI induced a significant increase in brain edema at 1, 3 and 5 days in the TBI group compared with the sham control. RV administered post-injury significantly reduced brain edema following TBI.

To determine the neuroprotective effects of RV against TBI-induced brain damage, the effects of RV pretreatment on the learning and memory ability (Fig. 2) and motor deficits (Fig. 3) were examined using the MWM and NSS score following TBI, respectively. As expected, TBI resulted in a significant deficit in spatial learning compared with the sham group, and the administration of RV significantly reduced the escape latency at 3, 4 and 5 days compared with the TBI group. In addition, the NSS of the rats in the TBI group were observed to significantly increase in comparison with the sham group at 1–4 days, and RV treatment significantly improved the motor function recovery of the TBI rats at 1–3 days following injury.

A number of studies have reported the significant loss of synapses in the days following brain injury, including in the brain regions connected to the site of initial injury, such as the hippocampus (1,4,24). Considering RV was able to improve spatial learning, it was examined whether RV treatment could affect synaptic alterations in the rat hippocampus following TBI. The expression of two synaptic proteins was evaluated in the rat hippocampus using western blot analysis at 1, 3 and 5 days following TBI. As demonstrated in Fig. 4A, there was a reduction in the expression levels of synaptophysin in the TBI group compared with the sham group at 1, 3 and 5 days. The reduction in the levels of synaptophysin indicated a loss of synapses in the TBI rats. The rats treated with RV exhibited significantly greater levels compared with the TBI group. In addition, Fig. 4B indicates that the levels of PSD-95 were reduced at 3 and 5 days following injury, with the RV treated rats significantly improved compared with the TBI group.

To determine how autophagic activity is altered following TBI, LC3 expression in hippocampal neurons at 24 h following TBI was detected using immunofluorescence. As shown in Fig. 5, LC3 staining is indicated in red, neurons stained with NeuN are in green, and DAPI staining of nuclei is in blue. The images were merged, and the co-localization of NeuN and LC3 was observed in the immunofluorescent staining at 24 h. The results indicated that alterations in LC3 proteins had occurred in neurons in the hippocampal region following TBI. In order to confirm the ability of RV to inhibit autophagy, the protein levels of LC3-II (ratio of LC3-II to LC3-I) and Beclin1 were determined by western blot analysis. The protein expression levels of LC3-II and Beclin1 in the hippocampus were significantly upregulated at 1, 3 and 5 days following TBI, with the highest level observed at 1 day (Fig. 6A). As the densitometry analysis indicates in Fig. 6B, treatment with RV significantly reduced the relative protein expression of LC3-II and Beclin1 at 1, 3 and 5 days in the hippocampus compared with the TBI group.