All experimental procedures were approved by The Third Military Medical University Committee (Chongqing, China) for the use and care of animals in research, and were performed in accordance with the The Third Military Medical University's guidelines for the care and use of laboratory animals. A total of 150 male Sprague-Dawley rats (age, 10–12 weeks; weighing, 280–320 g; obtained from The Third Military Medical University Animal Center) were used in the present study. All the rats had free access to food and water, and were housed under a standard 12-h light/dark cycle. The housing temperature was 25–28° and the rats were housed in groups.

A previously described TBI procedure (18) was performed in the present study. Briefly, the rats were anesthetized with sodium pentobarbital [i.p, 50 mg/kg; Bio-Rad Laboratories (Shanghai) Co., Ltd., Shanghai, China], and a 5-mm craniotomy was performed over the left parietal cortex, which was centered on the coronal suture [Bio-Rad Laboratories (Shanghai) Co., Ltd.] and 3 mm lateral to the sagittal suture. The TBI was performed using a pneumatic piston [Bio-Rad Laboratories (Shanghai) Co., Ltd.] with a rounded metal tip (2.5 mm diameter), which was angled 22.5° to vertical to ensure that the tip was perpendicular with the brain surface at the center of the craniotomy. A velocity of 4 m/s and a deformation depth of 2 mm below the dura were used to generate the TBI in the procedure. The bone flap was immediately replaced and sealed, and the scalp was closed using sutures. Body temperature was monitored throughout the surgery, to ensure it was maintained at 37.0±0.5°C. Whilst the rats were recovering from anesthesia, they were placed in a heated cage to maintain body temperature.

The rats were randomly divided into three groups: Sham-operated group (sham; n=15); the TBI group (TBI; n=35) and the TBI + harmine (Beijing Aoboxing Biotechnology Co., Ltd., Beijing, China)-treated group (Harmine; n=35). Harmine was administered immediately following TBI (i.p, 30 mg/kg per day) for up to 5 days. The sham and TBI groups received equal volumes of 0.9% saline solution (i.p.).

Brain edema was evaluated by analyzing the brain water content, as described previously (19). Following anesthetization, as described above, and sacrifice by exsanguination, the rat brains were separated and weighed immediately using a chemical balance, in order to obtain the wet weight (WW). Following drying in a desiccating oven for 24 h at 100°C, the dry tissues were weighed to obtain the dry weight (DW). The water percentage in the brain was calculated according to the following formula Brain water % = (WW − DW) / WW) × 100.

The neurobehavioral status of the rats was evaluated using a set of 10 tasks, collectively termed the neurologic severity score (NSS) (20), which assesses reflexes, alertness, coordination and motor abilities. A score of one is awarded for failure to perform a particular task; therefore, a score of 10 reflects maximal impairment, whereas a normal rat scores 0. The rats were grouped as follows for examination of behavioral recovery: Sham, n=3; TBI, n=7; and Harmine, n=7. Following TBI, the NSS was evaluated at 1, 3 and 5 days. Each rat was assessed by an observer who was blinded to the animal treatment. The difference between the initial NSS and that at any later time-point was calculated for each rat, and this value (ΔNSS) reflects the spontaneous or treatment-induced recovery of motor function. Furthermore, the spatial learning ability of the rats was assessed using a Morris water maze, as previously described (21). Briefly, the Morris water maze consisted of a black circular pool (diameter, 180 cm; height, 45 cm) filled with water (depth, 30 cm; temperature, 26°C) and divided into four equal quadrants: North (N), west (W), south (S) and east (E). An escape platform (diameter, 12 cm; height, 28 cm, made opaque with paint and submerged 2 cm) was placed in the center of one of the quadrants, equidistant from the sidewall and the center of the pool. Rats were trained to locate the platform prior to TBI or undergoing the sham surgery. For each trial, a rat was randomly placed into a quadrant start point (N, S, E or W) facing the wall of the pool and was allowed ≤60 sec to escape to the platform. Rats that failed to escape within 90 sec were placed on the platform for ≤20 sec and returned to the cage for a new trial (intertrial interval, 20 sec). The maze performance was recorded using a video camera suspended above the maze and interfaced with a video tracking system (HVS Imaging; Hampton, UK). The mean escape latency of a total of five trials was calculated. This assessment was performed 1, 3 and 5 days following TBI or sham surgery.

At 24 h post-surgery, the rats (sham, n=3; TBI, n=7; and Harmine, n=7) were anesthetized, as described above, and perfused intracardially with isotonic sodium chloride solution, followed by 4% (w/v) paraformaldehyde [Bio-Rad Laboratories (Shanghai) Co., Ltd.] in 0.1 M sodium phosphate buffer [pH 7.4; Bio-Rad Laboratories (Shanghai) Co., Ltd.]. The rats were sacrificed via exsanguination. The brains were subsequently removed and fixed for 48 h in 4% (w/v) paraformaldehyde. Following fixation, the brains were embedded in paraffin [Bio-Rad Laboratories (Shanghai) Co., Ltd.], sliced into 4 µm coronal sections at the bregma, and stained with H&E [Bio-Rad Laboratories (Shanghai) Co., Ltd.]. The surviving and dying neurons in the CA1 area per 1 mm were subsequently quantified under an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan).

At 24 h post-TBI, coronal sections were incubated with 10% normal donkey serum [Bio-Rad Laboratories (Shanghai) Co., Ltd. for 30 min at room temperature in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 [Bio-Rad Laboratories (Shanghai) Co., Ltd.]. The sections were then incubated at 4°C overnight with the following primary antibodies: Goat anti-rat neuron-specific nuclear protein [NeuN; 1:200 (cat. no. sc-31154); Santa Cruz Biotechnology, Inc., Dallas, TX, USA] and rabbit anti-rat caspase 3 [1:50; (cat. no. sc-7148); Santa Cruz Biotechnology, Inc.]. The sections were then washed with PBS four times at room temperature, followed by an incubation with appropriate fluorescent-labeled secondary antibodies [1:200 (cat. nos. sc-2342 and sc-2341) Santa Cruz Biotechnology, Inc.] for 1 h at room temperature. The sections were incubated with DAPI (1 ng/Nl; Beijing Aoboxing Biotechnology Co., Ltd.) to counterstain the nucleus. Subsequently, the sections were washed with PBS and mounted using water-based mounting medium containing anti-fading agents (Biomeda; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Confocal images were captured using a laser scanning confocal microscope (Olympus FV1000; Olympus Corporation, Tokyo, Japan) and digital imaging software (FV10-ASW 1.5 Viewer; Olympus Corporation).

The rats (sham, n=3; TBI, n=7; and Harmine, n=7) were anesthetized and underwent intracardiac perfusion with 0.1 mol/l PBS (pH 7.4). The cortex region of the brain was rapidly isolated and the brain tissues were homogenized using a homogenizer, total proteins were extracted using protein extraction reagent [both Bio-Rad Laboratories (Shanghai) Co., Ltd.] and protein concentration was determined using a bicinchoninic acid reagent (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) method. The protein samples (5 mg) were separated by 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis [Bio-Rad Laboratories (Shanghai) Co., Ltd.] and were subsequently transferred onto polyvinylidene fluoride membranes (Roche Diagnostics Deutschland GmbH, Mannheim, Germany). The blots were then blocked with 5% fat-free dry milk for 1 h at room temperature. Following blocking, the membranes were incubated with the following primary antibodies overnight at 4°C: Rabbit anti-GLT-1 polyclonal antibody (cat. no. sc-15317), rabbit anti-interleukin (IL)-1β polyclonal antibody (cat. no. sc-7884), rabbit anti-tumor necrosis factor (TNF)-α polyclonal antibody (cat. no. sc-7895), rabbit anti-caspase 3 polyclonal antibody (cat. no. sc-7148) and mouse anti-β-actin monoclonal antibody [(cat. no. sc-7210) all 1:500; Santa Cruz Biotechnology, Inc.). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; cat. no. sc-2027) and anti-mouse IgG (cat. no. sc-2025) for 2 h at room temperature (1:5,000; Santa Cruz Biotechnology, Inc.). Following incubation with the secondary antibody, the immunoblots were then visualized following development with an enhanced chemiluminescence detection system [Bio-Rad Laboratories (Shanghai) Co., Ltd.], and densitometric signals were quantified using an enhanced chemiluminescence detection system [Bio-Rad Laboratories (Shanghai) Co., Ltd.]. The western blotting results were analyzed using ImageJ 1.41 software (National Institutes of Health, Bethesda, MD, USA).

Data are expressed as the mean ± standard error of the mean. SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. Statistical analysis was performed using one-way analysis of variance followed by the Student-Newman-Keuls post-hoc 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, brain water content was significantly increased in the TBI group, compared with the sham group at 1, 3 and 5 days following trauma. Treatment with harmine significantly reduced the tissue water content at 1, 3 and 5 days, a compared with the TBI group.

Since treatment with harmine successfully attenuated brain edema, the present study examined whether harmine also improved spatial learning function, which was assessed using a Morris water maze at 1, 3 and 5 days post-TBI or sham surgery. As shown in Fig. 2, TBI resulted in a significant spatial learning deficit at 1, 3 and 5 days, compared with the sham group, and harmine treatment significantly reduced the escape latency at 3 and 5 days, compared with the TBI group.

Temporal variations in the functional recovery of the TBI rats are demonstrated in Fig. 3 and expressed as ΔNSS. Post-TBI administration of harmine significantly improved the motor function recovery of the rats at 1, 3 and 5 days following TBI, compared with the TBI group without harmine treatment.

Neuronal survival was assessed at 24 h using H&E staining. Morphologically, the nuclei of the normal neurons were round and poorly stained, whereas the nuclei of the dying neurons were pyknotic and darkly stained (Fig. 4A). As shown in Fig. 4B, compared with the sham group, TBI resulted in a marked decrease in the survival rate of the neurons. However, the neuronal survival rate in the harmine-treated group was significantly increased, compared with the TBI group.

The protein expression levels of GLT-1 in the hippocampus were analyzed using western blot analysis. As shown in Fig. 5, at 1, 3 and 5 days post-surgery, there was a significant downregulation in the expression of GLT-1 in the TBI group, compared with the sham group. Administration of harmine resulted in marked elevation in the expression of GLT-1, compared with the TBI group.

The expression levels of IL-1β and TNF-α were detected in the hippocampus 1, 3 and 5 days following TBI. As shown in Fig. 6, the expression levels of the inflammatory cytokines in the sham group were low; however, the expression levels of IL-1β and TNF-α were markedly elevated at each of the time-points in the TBI group. Furthermore, TBI-induced expression of IL-1β and TNF-α was significantly reduced following administration of harmine.

The co-localization of NeuN and caspase 3 was detected using immunofluorescent staining 24 h post-TBI. As shown in Fig. 7, the majority of TBI-induced apoptosis occurred in the neurons.

The expression of caspase 3 in the hippocampus were detected using western blot analysis. As shown in Fig. 8, at 1, 3 and 5 days post-TBI, the expression of caspase 3 was significantly increased in the TBI group, compared with the sham group. However, the administration of harmine significantly reduced the expression of caspase 3, compared with the TBI group.