Contributed equally
The present study compared the potential neuroprotective effect of tanshinone IIA (TIIA) monotherapy, methylprednisolone (MP) monotherapy and combined treatment in an adult acute spinal cord injury (ASCI) rat model. The current study used the weight-drop method (Allen's Impactor) in the rat model and the mechanical scratch method in primary spinal cord neuron culture to determine whether the combined treatment was able to reduce the required dosage of MP in the treatment of ASCI to produce a similar or improved therapeutic effect.
Acute spinal cord injury (ASCI) is a severe type of high-energy central nervous system (CNS) damage that frequently leads to devastating neurological deficits and disabilities, including the loss of sensory and motor capabilities (paraplegia or tetraplegia). The prevalence of ASCI ranges from 906 to 250 per million and the annual incidence of ASCI ranges from 8 to 49.1 per million in different nations (
Tanshinone IIA (TIIA) is an effective monomer component purified from the Chinese herb Danshen (
All Sprague-Dawley rats were kept in dams in the Laboratory Animal Services Center with
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 (
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 (
Primary culture of cortical neurons was isolated and cultured as previously described (
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 ×
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) (
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 (
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 (
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
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;
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 (
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 (
Immunofluorescence double labeling images are presented in
The present study provides evidence that the combination of TIIA with MP significantly improves motor function following ASCI and may reduce the dose of MP required to induce the same anti-apoptosis, antioxidation and anti-inflammatory efficiency. Furthermore, combined treatment may reduce apoptosis of neurons following mechanical injury
The treatment of ASCI is a major challenge for physicians due to the complex and progressive nature of the disease. MP is one of the most investigated agents due to its neuroprotective potential and remains the only drug used worldwide to treat ASCI. Although MP is not FDA-approved for use following ASCI, it is commonly used in this setting based on the results of a number of randomized controlled trials; furthermore, the beneficial effect of high-dose MP was initially reported in a series of National Acute Spinal Cord Injury Studies (NASCIS) (
When the spinal cord is lacerated or macerated by a sharp penetrating force, or contused or compressed by a blunt force, it leads to neurological damage occurring in the spinal cord that is normally referred to as ‘primary injury’. The primary injury refers to the loss of spinal cord integrity due to mechanical factors (
Assessment of neurological function is a prevalent method of accessing the degree of injury and the outcome of a treatment with medication. In the present study, BBB rating indicated a significant improvement of locomotion occurring everyday post-treatment, suggesting that the combined treatment may improve the behavioral function in animals with ASCI. Notably, the BBB scores in the MP group were higher than the combined treatment group 3 days following ASCI, while functional recovery was slower, taking 4–7 days. This may be because high-dose MP is more effective in protecting the injured spinal cord in the early phase, while continuous TIIA treatment may protect the injured spinal cord in the late phase.
The potential mechanism of ASCI remains unclear. However, neuronal apoptosis is one of the major pathogenic mechanisms that govern ASCI. The increased activation of oxidative stress and a series of apoptosis-associated proteins contribute to the pathogenic processes of neuronal death following ASCI, which ultimately lead to cell death (
In the present study, to investigate the protective effects of a combination treatment with TIIA and MP on the ASCI model, the factors associated with apoptosis were investigated. The data indicated that a combined treatment may markedly reverse the increase in pro-apoptosis factors Bax and caspase-3, and the decreased anti-apoptosis factor Bcl-2 protein induced by ASCI
Secondary injuries develop hours to days following primary injuries and involve a number of pathophysiological changes including ischemia, ion infiltration, production of oxygen free radicals and lipid peroxidation (
NF-κB was key to the inflammatory action due to its regulatory role on the expression of a variety of cytokines (such as TNF-α and IL-6) that regulate the inflammatory response (
In conclusion, the present study demonstrates that TIIA combined with a low dose of MP may protect the spinal cord from ASCI due to its antioxidant, anti-inflammatory and anti-apoptosis properties in the spinal cord tissue following ASCI. The current study, to the best of our knowledge, is the first to reveal that TIIA combined with low dose MP therapy has the equivalent efficiency of a full dose of MP monotherapy
The authors would like to thank Dr. Hui Kang (Wuhan General Hospital of Guangzhou Military Command, Wuhan, China) for his advice about the experiment design and Professor Yi Zhang (Wuhan General Hospital of Guangzhou Military Command, Wuhan, China) for his technical assistance and his assistance with rat husbandry.
Effects of each group on functional recovery following ASCI by BBB scores. *P<0.05 vs. all treatment groups; **P<0.05 vs. MP group. ASCI, acute spinal cord injury; BBB, Basso Beattie Bresnahan; MP, methylprednisolone; TIIA, tanshinone IIA; TIIA + MP, TIIA and MP combined treatment; Sham, sham-operated, no treatment.
Expression of Bcl-2, Bax and Caspase-3 proteins determined by western blot analysis. (A) Results normalized relative to β-actin in the ASCI rats at day 7 and (B) in spinal neuron culture scratch models following 24 h culture. Data are presented as mean ± standard error of the mean, n=4 per group. (A) *P<0.05 vs. ACSI group; #P<0.05 vs. TIIA + MP group. (B) *P<0.05 vs. ASCI group; #P<0.05 vs. TIIA + MP group and MP group. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated protein X; ASCI, acute spinal cord injury; MP, methylprednisolone; TIIA, tanshinone IIA; TIIA + MP, TIIA and MP combined treatment; Sham, sham-operated, no treatment.
Expression of antioxidation molecules. The expression of (A) SOD and (B) MDA were determined at day 1 and 7 in ASCI rats. Data are presented as mean ± standard error of the mean, n=4 per group. (A) *P<0.05 vs. ASCI group; #P>0.05 vs. TIIA + MP group. (B) *P<0.05 vs. ASCI group; #P>0.05 vs. TIIA + MP group. SOD, superoxide dismutase; MDA, malondialdehyde; ASCI, acute spinal cord injury; MP, methylprednisolone; TIIA, tanshinone IIA; TIIA + MP, TIIA and MP combined treatment; Sham, sham-operated, no treatment.
Expression of NF-κB determined by western blot analysis, with results normalized relative to β-actin in spinal neurons culture scratch models following 24 h. Data are presented as mean ± standard error of the mean, n=4 per group. *P<0.05 vs. ASCI group, #P>0.05 vs. TIIA + MP group. NF-κB, nuclear factor-κB; ASCI, acute spinal cord injury; MP, methylprednisolone; TIIA, tanshinone IIA; TIIA + MP, TIIA and MP combined treatment; Sham, sham-operated, no treatment.
Activity of neurons to assess and select the concentration of TIIA required for subsequent experiments. (A) *P<0.05 vs. unmarked groups; #P>0.05 vs. 10 µM group. (B) *P<0.05 vs. ASCI group; #P<0.05 vs. TIIA + MP group; **P>0.05 vs. TIIA + MP group. TIIA, tanshinone IIA; ASCI, acute spinal cord injury; MP, methylprednisolone; TIIA + MP, TIIA and MP combined treatment; OD, optical density.
Immunofluorescence double labeling images and the R value of Mander's overlap coefficient indicate that the immunoreactivity of capase-3 increases in ASCI rats and was reduced following treatment with MP. Scale bar=1 µM. (A) Expression of caspase-3 in the spinal tissues and neurons