Animals and Experimental Design
Adult male Sprague–Dawley rats, weighing 220–250 g, were obtained from the Center of Experimental Animals, Central South University. All surgical interventions, perioperative care and drug administrations were performed in accordance with internationally accepted principles, and were approved by the Animal Use and Ethics Committee of Central South University. The rats were housed three (two after SCI modeling) per laboratory cage, with free access to food and water, in identical environments (temperature 22–24 °C; humidity 60–80 %). The experimental rats were randomly divided into the following three groups: Sham group, normal saline (NS) group and TMP group. Each group was divided into 6 subgroups (n = 10 per subgroup) according to the time of sacrifice: 1, 3, 7, 14, 21 and 28 days post-injury (dpi).
Contusive Spinal Cord Injury Modeling
The establishment of contusive SCI model was performed as previously described , with slight modification. Briefly, the rats were intraperitoneally (i.p.) anesthetized with chloral hydrate, following which they received conventional skin preparation and antisepsis. Laminectomy was performed at thoracic vertebra level 10 (T10) in all groups, followed by contusive SCI of moderate severity (8 g weight × 3 cm vertical height free falling) in the NS and TMP groups. Successful signs of contusive SCI modeling were as follows: bleeding and edema in the T10 spinal cord, retracted flapping of both hind limbs, spasmodic sway of the tail, and flaccid paralysis after palinesthesia.
TMP was purchased from Tianjin Jinyao Amino Acid Co. Ltd., China. There were some differences in the effective dosage of TMP among other published reports [14, 18]. Taking the therapeutic and side effects into consideration, we ultimately chose 80 mg/kg as the application dosage in this study. TMP was injected i.p. daily for 5 consecutive days from day 3 post-injury in the TMP group, which was replaced by equal volumes of normal saline in the NS group.
Behavioral assessments were performed prior to surgery and at 1, 3, 7, 14, 21 and 28 days post-injury in all three groups, based on a 21-point Basso–Beattie–Bresnahan (BBB) Locomotor Rating Scale, where 0 reflects no locomotion and 21 reflects normal motor functions . Hind limb movements, trunk position and stability, tail position, stepping, coordination, paw placement and toe stretching were all observed over a 5 min period for each rat, by two independent examiners who were blinded to the experimental design.
Spinal cord tissues were mainly harvested by two methods. The first method involved direct separation of about 1 cm length of spinal cord tissue at T10, after the rats were euthanized. The removed tissues were then immediately preserved in liquid nitrogen. These tissues were used for total RNA and protein extraction. The second method was as follows: the rats were deeply anesthetized and perfused with 300 ml 37 °C heparinized normal saline, through the ascending aorta, followed by 4 % paraformaldehyde (PFA) in 0.1 M PBS; the spinal cord tissues around T10 were then carefully removed, post-fixed in 4 % PFA overnight at 4 °C; serially dehydrated in 15 and 30 % sucrose solution until sinking, the dehydrated tissues were thereafter embedded in optimal cutting temperature (O.C.T.) compound, and frozen at −20 °C. The frozen O.C.T.-embedded tissues were sectioned at a 10-μm thickness for immunofluorescence, TUNEL and Nissl assays.
Quantitative Real-Time PCR Analysis
Total RNA was extracted using the Trizol method, according to the manufacturer’s instructions (Invitrogen, USA). After reverse transcription was performed, quantitative real-time PCR (qRT-PCR) was analyzed in a BIO-RAD CFX96 Real-Time System, using SYBR® Premix Ex Taq™ II (Takara, Japan). The primers were designed using NCBI primer-blast and synthesized by Sangon biotechnology (Shanghai, China) as follows: PGC-1α (forward: GGA CAC GAG GAA AGG AAG ACT A; reverse: GTA GCA CTG GCT TGA ATC TGT G) and β-actin (forward: CCC ATC TAT GAG GGT TAC GC; reverse: TTT AAT GTC ACG CAC GAT TTC). The specificity of PCR products was guaranteed by melting curve analysis. The expression of PGC-1α was normalized to β-actin, and the gene expression was comparatively analyzed using the 2−ΔΔCt method.
Western Blot Analysis
Total proteins were extracted using RIPA buffer and protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, China). Protein concentrations were determined using the BCA method. Then, equal amounts of protein extracts were separated in an 8 % gel by SDS-PAGE, followed by transfer onto a polyvinylidene fluoride (PVDF) membrane. Blocking was performed for 1 h with 5 % skim milk in 0.05 % Tween-20 in PBS (PBST) at room temperature. Thereafter, the PVDF membrane was incubated with the primary antibody in 5 % IgG-free BSA (Genview, USA) overnight at 4 °C, followed by incubation with the horseradish peroxidase-conjugated secondary antibody for 1 h, after rinsing in PBST. Then, the membrane was rinsed in washing buffer and the signal was detected with enhanced chemiluminescence. Primary antibodies included PGC-1α (Novus, USA; 1:1500) and β-actin (BBI, China; 1:1000). Band intensities were quantified using Image J software. Values were normalized to β-actin in all samples.
Immunofluorescence staining was performed on frozen sections. In brief, sections were rinsed in PBS and treated with 0.3 % Triton X-100 in PBS to disrupt the membranes. After blocking with 10 % normal goat serum for 1 h to reduce nonspecific staining, sections were incubated with the primary antibodies, mouse anti-beta III tubulin (Abcam, USA, 1:100) and rabbit anti-PGC-1α (Novus, USA, 1:150) overnight at 4 °C for 24 h. After rinsing in PBS, the secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG (H + L) (Jackson, USA, 1:600) and Cy3 goat anti-rabbit IgG (H + L) (Jackson, USA, 1:800), were added onto sections in 5 % blocking solution, to incubate at room temperature for 30 min. Then, the sections were counterstained with DAPI (Sigma, USA), and imaged using a Leica DFC310 FX microscope (Leica, Japan).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed on frozen sections, to detect apoptotic changes of neural cells after contusive SCI, using the TUNEL staining apoptosis detecting kit (Nanjing KeyGen Biotech Co. Ltd., China) according to the manufacturer’s protocol. To reveal total cells, we counterstained the same sections with hematoxylin. The number of TUNEL-positive and total neural cells was counted in five different microscopic fields per section, and the TUNEL-positive percentage was calculated.
Nissl staining was performed on frozen sections 5 mm rostrally to the lesion epicenter, with Nissl solution (0.1 % Cresyl violet) for 20 min. After rinsing in distilled water, the sections were differentiated in 95 % ethyl alcohol. Then, the sections were rinsed in 70 % ethyl alcohol, dehydrated in increasing concentrations of ethyl alcohol, and cleared in xylene. The cells containing Nissl bodies were considered as neurons, and those with typical neuronal morphology were counted per section. One out of every two sections was taken and a total of five sections were counted for each group to determine neuronal survival. A typical neuron exhibited a large amount of granule-like dense bluish violet staining indicating Nissl bodies in a regularly polygonal cell body. In a damaged neuron, Nissl bodies decreased in number and density, and the cell body became relatively small and irregular.
Statistical analysis was performed using SPSS 19.0 software (IBM, USA). Data were presented as the mean ± standard deviation (SD). The statistical significance of differences was determined using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test for multiple comparisons, or Student’s unpaired t test between two groups. All analyses were based on at least three different experiments with duplicate samples. A value of P < 0.05 was considered to be statistically significant.