All Sprague-Dawley rats were kept in dams in the Laboratory Animal Services Center with ad libitum access to food and water and a 12-h light-dark cycle. All rats were maintained at 22–25°C and 40–60% humidity. A total of 60 adult male Sprague-Dawley rats (Hubei Provincial Laboratory Animal Public Service Center, Hubei, China), weighing 240–260 g and aged 6–7 weeks-old, were used in the current study, 12 of which were selected for sham-surgery and 48 selected for contusive ASCI. A total of 80 one-day-old male Sprague-Dawley rats (Center for Animal Experiment of Wuhan University, Wuhan, China) were used for cell culture in vitro. All rats were maintained in the same conditions. All procedures were performed in accordance with guidelines approved by the Animal Ethics Committee of Wuhan General Hospital of Guangzhou Military Command (Wuhan, China).

All adult animals (n=60) were anaesthetized through intraperitoneal (i.p.) injection of 10% chloral hydrate (3.5 ml/kg, i.p.; Hubei Yuancheng Saichuang Technology Co., Ltd., Wuhan, China). In order to expose the paravertebral muscles, an incision was made along the back of each rat. A laminectomy was performed at the T9-T11 level, exposing the cord without damaging the dura. In order to subject the exposed dorsal surface of the cord to the weight drop impact, a 10-g metal rod was dropped from a height of 25 mm (19). Subsequently, the lesioned muscle and skin were sutured in two layers. Each rat was then administered 1.0 ml saline solution using a subcutaneous injection, to replace blood volume lost during surgery. Following surgery, sterile gauze was wrapped around the wound on each rat. A standard diet and water were provided daily to the rats ad libitum. A total of 3 to 5 days' post-injury, manual bladder expression was performed twice a day until the rats were able to urinate by themselves.

In the current study, 60 rats were randomly assigned into five groups (n=12, each group) and all animals were subjected to laminectomy: Sham group, animals were only subjected to laminectomy; ASCI group, animals subjected to ASCI using an impactor; TIIA treatment group, animals subjected to ASCI using an impactor and treated with a dosage of 30 mg/kg TIIA per day [chosen according to previous findings (20)]; MP treatment group, animals subjected to ASCI using an impactor and treated with 30 mg/kg MP (13) 15 min following the strike; TIIA + MP treatment group, the animals were subjected to ASCI using an impactor and treated with 30 mg/kg TIIA per day and a selected low dose of 20 mg/kg MP 15 min after the strike. The drugs were administered via intraperitoneal administration. At the planed time point, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5 ml/kg, i.p.) as before and the spinal cord was immediately exposed from T9 to T11 and 10 mm damaged tissue was cut at the site of injury. Rats were sacrificed by cervical dislocation except for the immunofluorescence double labeling experiment at different time points (4 rats per group), depending on the parameters measured: Rats undergoing western blot analysis for nuclear factor (NF)-κB on day 1, measurement of tissue superoxide dismutase (SOD) and malondialdehyde (MDA) on day 1 and on day 7 following ASCI, immunofluorescent staining on day 3 following ASCI and western blot analysis for caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated protein X (Bax) on day 7 following ASCI.

Primary culture of cortical neurons was isolated and cultured as previously described (21), with minor modifications. For mixed glial cell culture, one-day-old Sprague-Dawley rat pups were sacrificed by cervical dislocation; the skin and skull were carefully torn with tweezers and brain cortical tissues were dissected in ice-cold dissection buffer. Following this, the meninges of the cortex were peeled off and separated from the olfactory bulb and hippocampus and dissected cortical tissues were digested with trypsin. Following incubation at 37°C for 15 min to adhere and eliminate fibroblasts, cells were dissociated and plated into 75 cm² tissue culture flasks at a density of 2.0×10⁷ cells/flask. Cells were incubated at 37°C with Dulbecco's modified Eagle's medium/F12 supplemented with 10% fetal bovine serum (both Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin-streptomycin medium. Following 24 h, non-adherent cells were removed by agitating the flask and the culture medium was replaced. When a confluent layer of glial cells formed (10 to 14 days), cells were trypsinized (Beyotime Institute of Biotechnology, Haimen, China) and inoculated. Following the second passage, cells were seeded into a 6-well cell culture plate (Nest Biotechnology Co., Ltd., Wuxi, China) for neuron-glia co-culture. Rat primary spinal cord neuron cultures were derived from the spinal cord of one-day-old Sprague-Dawley rat pups. Briefly, spinal cords were dissected and freed of meninges. Cells were dissociated by trypsinization, followed by triturating and passing through a 74-mm steel mesh. Cells were incubated at 37°C for 30 min to allow adherence and eliminate glial cells and fibroblasts. Neurons (2×10⁵ cells/plate) were plated onto poly-L-lysine (PLL; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) coated 20-mm glass coverslips (NEST, China) for co-culture experiments and MTT analysis. Neurons were maintained in neurobasal media (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2% B27 supplement (Gibco; Thermo Fisher Scientific, Inc.) and 0.5 mM glutamine (Sigma-Aldrich; Merck Millipore). Neurons on coverslips at day 6–7 were placed into glia-seeded wells. Neurons and astrocytes were in close apposition but with no direct cell-cell contact. For modelling ASCI, neurons and glial cell were subjected to a cut with a no. 15 scalpel blade (22) with a 1 mm gap. Cultures were maintained in a 37°C humidified incubator in a 5% CO2 atmosphere. The cells were treated with TIIA at a concentration of 10 µM (according to the MTT analysis), MP at a concentration of 1 µM and combined TIIA 10 µM and MP 0.5 µM, respectively. The incubation was continued for 24 h and subsequently the cells were trypsinized, collected and extracted for analysis.

The neurons' viability was measured by MTT assay for the optimal concentration of TIIA for neurons. Briefly, the MTT solution (Beyotime Institute of Biotechnology) was added to 96-well plates (1×10⁴ neuronal cells/well) at a final concentration of 500 mg/ml and then incubated for 4 h at 37°C. To dissolve the formazan, the culture medium was removed and 100 µl dimethyl sulfoxide was added to each well. The optical density of each well was measured at 570 nm using a microplate reader. One culture well with neurons was used for each experimental condition. Thus, each plate contained multiple wells of each experimental condition and multiple control wells. This procedure was replicated for 4 plates per condition. Spinal cord neurons were treated with various concentrations (0, 0.1, 0.5, 1, 5, 10, 30, 50, 70 and 90 µM) of TIIA for 24 h. Following this, the cells were injured as previously described. Cell viability was measured using an MTT assay. Neuron viability treated with TIIA, MP and their combination was measured by MTT assay too. The MTT data was converted to the percentage of the respective control groups (untreated spinal cord neuron cells) after analysis by microplate reader (SM-600, Shanghai Utrao Medical Devices Co., Ltd, China).

Total proteins were extracted separately from the spinal cord tissues or neurons' cultures. In detail, following sacrifice and transcardial perfusion with 100 ml 0.9% saline, the experimental rats' skin and soft tissue on back at T9-T11 were cut to expose the injured spinal cord, which was harvested (5 mm rostral and 5 mm caudal from the injury site). The tissue was homogenized by sonication in radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China), and neurons were washed three times in Earle's Balance Salt Solution following trypsinization and kept in serum-free media (Hyclone; GE Healthcare Life Sciences). The samples were centrifuged at 12,000 × g for 1 h at 4°C. The protein content of the lysate samples was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Protein lysates (35 µg per lane for each sample) were fractioned using 10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were then blocked with 5% non-fat dry milk at room temperature for 2 h and incubated for 12 h at 4°C with primary antibodies: Rabbit anti-β-actin (BA2305; 1:1,000; Wuhan Boster Biological Technology, Ltd.), rabbit anti-Bcl-2 (15071T; 1:800), rabbit anti-Bax (2772T; 1:800; both Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-caspase-3 (ab4051; 1:300; Abcam, Cambridge, UK) and rabbit anti-NF-κBP65 (BA1298; 1:1,000; Wuhan Boster Biological Technology, Ltd.). Horseradish peroxidase conjugate goat anti-rabbit immunoglobulin (Ig)G was used as a secondary antibody (BA1055 1:50,000, Wuhan Boster Biological Technology, Ltd.). The reaction was developed using enhanced chemiluminescence (ELC) reagent (Thermo Fisher Scientific, Inc.) and signal density was measured using ImageJ analysis software v2.1.4.7 (National Institute of Health, Bethesda, MD, USA) (23). Each group had 4 replicates and the values were normalized against β-actin.

In order to perform immunofluorescence double labeling for confocal microscopy, rats were sacrificed 3 days post-injury as described before, and the whole body of the rat was fixed using transcardial saline infusion followed by 100 ml paraformaldehyde (4%). Following perfusion, the injured spinal cords were carefully dissected, as indicated for western blot analysis, fixed for an additional 2 h in 4% paraformaldehyde at 4°C. The specimens were transferred to a solution containing 30% sucrose in 0.1 M phosphate buffer (pH 7.4) overnight. Spinal cord segments from sham-operated or injured animals were embedded in paraffin and longitudinally sectioned (2-µm thick), prior to mounting on gelatin-coated slides. The sections were dewaxed with xylene, permeabilized and blocked with 0.3% Triton X-100 and 10% normal goat serum (Wuhan Boster Biological Technology, Ltd.) in 0.01 M phosphate-buffered saline for 30 min at 4°C. Mouse anti rat caspase-3 polyclonal antibody (AB208161; 1:100; Abcam) and rabbit anti-rat β-III Tubulin (AT809; 1:100; Beyotime Institute of Biotechnology) primary antibodies were applied to the sections overnight at 4°C. On the following day, the sections were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit (BA1105; 1:1,000) and indoles cyanine dye (Cy3)-conjugated goat anti-mouse (BA1032; 1:1,000; both Wuhan Boster Biological Technology, Ltd.) secondary antibodies at 4°C for 3 h. Slides were mounted and examined with a BX51 fluorescence microscope (Olympus Corp., Tokyo, Japan). In control sections, the primary antibody was substituted with 1% normal goat serum. The colocalisation area was measured using ImageJ analysis software (v2.1.4.7). R value represented Mander's overlap coefficient tubulin and caspase-3 in ImageJ analysis software: Ranges between 1 and 0, with 1 indicating high colocalisation and 0 indicating low colocalisation.

Behavioral assessments were determined using the Basso, Beattie, and Bresnahan (BBB) locomotor rating score. Gross BBB locomotor recovery following contusive spinal cord injury was scored in an open field according to the locomotor rating scale of 0 (complete paralysis) to 21 (normal locomotion) (24). BBB testing was performed once a day in double-blind model from day 0 to day 7 following spinal cord surgery. Each rat was observed for 4 min by two investigators blinded to the study.

MDA was measured using an MDA assay kit (Thiobar bituric acid test method; Beyotime Institute of Biotechnology) and SOD activities in spinal cord tissues were measured using a Total-SOD (T-SOD) assay kit (Hydroxylamine method; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with previous research (20).

Data are expressed as the mean ± standard error of the mean. Statistical significance was determined using Student's t-test when there were two experimental groups. When more than two groups were compared, statistical evaluation of the data was performed using one-way analysis of variance and Dunnett's post-hoc test. P<0.05 was considered to represent a statistically significant difference.

In order to assess the effects of treatment on functional recovery following ASCI in each group, the BBB locomotor test was performed every day for 7 days (Fig. 1). On day 0, the BBB score of all rats was regarded as 0. BBB scores in the treatment groups were significantly higher than the ASCI group on day 3 and 7 (P<0.05). BBB scores in the TIIA + MP combined group were higher than the MP group on day 3, but this was not significant (P>0.05). From day 5 to day 7, the scores in the combined treatment group were higher than the MP group and the differences were statistically significant (P<0.05; Fig. 1).

In the present study, the effect of each treatment on the expression of pro- and anti-apoptotic factors on day 7 following ASCI in rats and 24 h following mechanical injury in neurons were investigated using western blot analysis. As presented in Fig. 2A, Bax and caspase-3 expression were appreciably increased in the spinal cords of rats subjected to ASCI and Bcl-2 expression was decreased compared with the sham group. However, this state was altered by the drug treatments. Combined treatment exhibited significant anti-apoptotic ability compared with that observed in the ASCI group on day 7 after ASCI (P<0.05; Fig. 2A). The statistical analysis demonstrated that Bcl-2 expression in the combined treatment group was significantly increased, however, levels of Bax and caspase-3 were significantly decreased compared with the MP group (P<0.05; Fig. 2A). In addition, the data in Fig. 2B indicated that combined treatment was significantly more effective than TIIA treatment alone at reversing the decrease in Bcl-2 expression induced by mechanical injury in neurons (P<0.05), although Bax and caspase-3 expression did not differ significantly. These effects were similar to those observed in the MP group (P<0.05).

The effect of each treatment on antioxidation was investigated by measuring the tissue levels of SOD and MDA in the spinal cord on day 1 and day 7. Levels of SOD were significantly elevated in the MP group and TIIA + MP group compared with the ASCI group (P<0.05; Fig. 3A), while levels of MDA were significantly reduced in the MP group and TIIA + MP group compared with those of the rats in the ASCI group (P<0.05; Fig. 3B). However, no significant differences in SOD and MDA levels were observed between the MP and combined treatment groups.

The effect of each treatment on NF-κB expression in the spinal cord tissue on 1 day following ASCI was detected by western blot analysis. This was due to the NF-κB associated pathway that serves a key role in inflammation during secondary injury following ASCI (25). Substantial increases in NF-κB levels were identified in spinal cord tissue samples collected from rats 1 day following ASCI. These increases were significantly reversed in all treatment groups (P<0.05; Fig. 4). NF-κB levels detected in the combined treatment group were lower than in the MP group, but this difference was not significant.

To determine the neuroprotective ability of TIIA, cultured spinal cord neurons were exposed to mechanical injury and cell viability was assessed by MTT reduction assay after 24 h. Results are presented as the mean of 5 replicate values in three independent experiments. Based on the result, 10 µM was selected as the optimal TIIA concentration for subsequent experiments, as the increase in neuronal activity was greatest following treatment with 10 µM TIIA (Fig. 5A). In the ASCI group, the optical density (OD) value decreased 24 h after the mechanical scratch protocol. Treatment with TIIA, MP and the combined treatment significantly reversed this decrease in OD value (P<0.05). Cell viability in the combined treatment group was higher than in the MP group, however, no significant differences were observed in cell viability between cells treated with MP alone and the combined treatment.

Immunofluorescence double labeling images are presented in Fig. 6A (red: Tubulin and green: caspase-3). The results indicated that decreased levels of caspase-3 expression in neurons were exhibited in treatment groups when compared with the ASCI group. In Fig. 6B, the R values represent the overlap coefficient of tubulin and caspase-3 and the results from the immunohistochemistry experiments revealed that a significant increase in the immunoreactivity of caspase-3 was observed in ASCI rats compared with the sham group (P<0.05), which suggested that there was more caspase-3 present in neurons in the ASCI group compared with the sham group. Similar to the results from western blot analysis, TIIA, MP and combined TIIA + MP treatment significantly reduced the R value compared with the ASCI group, which indicated that reduced levels of caspase-3 (P<0.05) were present in the neurons of each group, but there was no significant difference between the TIIA or MP groups compared with the combined treatment group (P>0.05).