Animals
Adult male Sprague-Dawley (SD) rats, weighing 180-220 g, were provided by the Center of Experimental Animals, Nantong University. All animal experiments were approved by the Animal Care and Use Committee of Nantong University and the Animal Care Ethics Committee of Jiangsu Province. All rats were housed in standard cages (five rats in each cage) in an environment maintained at 22±2℃ on a 12-12 h light-dark cycle and had free access to water and food.
Establishment of contusion SCI rat model and drug treatment
The number of animals subjected to surgery was calculated by six per experimental group in triplicate. The contusion SCI rat model was prepared as the previous description. In a nutshell, all animals were anesthetized with 10% chloral hydrate (3 mg/kg) administered intraperitoneally. The fur around the surgical site was shaved and the skin was disinfected with iodophor. Then the spinous processes of T8–T10 vertebrae were surgically exposed, and a laminectomy was performed at the ninth thoracic vertebral level (T9) with the dura remaining intact. The exposed spinal cord segment (about 3 mm in length) received a 150-kilodyne contusion injury using the IH-0400 Impactor (Precision Systems and Instrumentation) injury device. The impact rod was removed immediately, and the wound was irrigated. For drug delivery, 8 μl of 100 mM MIF inhibitor 4-IPP (TOCRIS) or D-DT inhibitor 4-CPPC (AOBIOUS) were slowly injected intrathecally, prior to the incision suture. The rats were subcutaneously administered with 0.2 ml antibiotics following surgery. Manual expression of bladders was performed twice a day until animals recovered spontaneous voiding.
Cell culture
Astrocytes were prepared from the spinal cord of newborn SD rats, 1-2 days after birth, and the astrocytes were isolated and cultured according to previously described methods. Briefly, the spinal cords removed from the spinal canal were placed into 0.01M PBS containing 1% penicillin-streptomycin. The spinal cord capsule was stripped clean under the microscope, following by minced with scissors and digested with 0.25% trypsin for 15 minutes at 37℃. Digestion was terminated by addition of Dulbecco’s Modified Eagle’s Medium - high glucose medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-glutamine. The suspension was then centrifuged at 1200 rpm for 5 minutes and the cells were resuspended and seeded onto poly-L-lysine pre-coated culture flask in the presence of 5% CO2. The medium was changed every 3 days until the whole flask is covered with cells. After 7-9 days, the culture flask was shaken at 250 rpm overnight to remove non-astrocytes. Astrocyte phenotype was evaluated by cell exhibiting a characteristic morphology and positive staining for the astrocytic marker glial fibrillary acid protein (GFAP). Astrocytes with purity more than 95% are acceptable for subsequent experiments.
Western blot
Protein was harvested from cells with a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1 mM PMSF, following treatment with recombinant rat D-DT protein (Aviva Systems Biology) for 24 h. Alternatively, protein was extracted from 1 cm spinal segments of injured site at 0 day, 1 day, 4 days, and 1 week following contusion (n = 6 in each time point). Samples were vortexed for 30 minutes and centrifuged at 12000 rpm for 15 minutes. The supernatants were collected and stored at -20 ℃ for use. Protein concentration of each specimen was measured by the BCA method to maintain the same loads according to the manufacturer’s instructions. Proteins were heated at 95 ℃ for 5 minutes, and 20 μg of each sample were electrophoretically separated on 10% SDS-PAGE gel, followed by transferring onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and then an overnight incubation at 4 ℃ with primary antibodies: D-DT (1:500, Abcam); COX1 (1:1000, CST); COX2 (1:1000, Cayman); mPGES-1 (1:200, Cayman); mPGES-2 (1:200, Cayman); cPGES (1:1000, Abcam); CD74 (1:1000, Biorbyt). After washing 3 times with TBST for 10 min each, the membrane was incubated with secondary antibody goat-anti-mouse HRP or goat-anti-rabbit HRP (1:1000, Beyotime) for 2 h at room temperature. The HRP activity was detected using an ECL kit. The image was scanned with a GS800 Densitometer Scanner (Bio-Rad), and the data were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-actin (1:5000) was used as an internal control.
ELISA
Cells or tissue samples were sonicated using the lysis buffer supplemented with a protease inhibitor PMSF as mentioned above. Homogenate was centrifuged at 12000 rpm for 15 min at 4 ℃, and the supernatant was collected for PGE2 ELISA assay (ARBOR ASSAYS) according to the manufacturer’s directions. The concentrations of PGE2 are expressed as pg/ml. Plates were read with a multifunctional enzyme marker (Biotek Synergy2) at a 450 nm wavelength.
Tissue immunofluorescence
The vertebra segments were harvested from six experimental models of each time point, post-fixed, and sectioned. The sections were blocked with 0.01 M PBS containing 3% BSA, 0.1% Triton X-100 and 10% normal goat serum for 1 h at 37℃, and incubated overnight at 4℃ with primary antibodies: GFAP (1:400, Sigma); OX42 (1:200, Abcam); MBP (1:500, CST); NeuN (1:1000, Abcam); D-DT (1:200, Abcam); S100β (1:400, Abcam); COX2 (1:200, Cayman); CD74 (1:50, Bioss). Thereafter, the sections were rinsed with PBS and incubated with the Cy3-labeled goat anti-rabbit IgG (1:400, Proteintech) or the Alexa Fluor 488-labeled donkey anti-mouse IgG (1:400, Abcam). Sections were observed under a fluorescence microscope (ZAISS, axio image M2).
Behavioral tests
The hindlimb locomotor function recovery was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor scale as previously described (Zhou et al., 2018). Breifly, after intrathecal injection of 4-CPPC or vehicle at 0, 7, 14, and 21 days, three well-trained investigators blind to the study were invited to observe the behavior of rats for 5 min. The BBB score ranged from 0 to 21 according to the rating scale. Every rat had a BBB score of 21 before surgery, and 0 to 1 after a successful SCI.
Statistical analysis
Statistical analysis used GraphPad Prism 8 software (San Diego, CA, USA). Normality and homoscedasticity of the data were performed using Levene’s test. Independent sample t-test and One-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc comparisons tests were utilized for comparisons for different groups. All data were presented as mean ± standard deviation (M ± SD). Two-sided p value < 0.05 was considered statistically significant.