SCI model
Adult female C57 mouse (age between 6-8 weeks) were purchased from specific pathogen-free (SPF) animals center in Shenzhen, China. All animal procedures were as the Animal Care guidelines and approved by Ethics Committee of First Affiliated Hospital of Southern Science and Technology University (Shenzhen People’s Hospital). The mice were chew and housed in a SPF level laboratory with standard 12-h light/dark cycles. In brief, mice were anesthesia with pentobarbital sodium (40mg/kg, i.p.). We incised the skin in the midline, laminectomy and complete spinal cord transection was performed at the thoracic 8/9 level according to previous study[19]. After procedure, the muscles and skin were sutured in layers.
Treatment Group
PTPσ intracellular sigma peptide (ISP) purchased from Creative Biomart (Shirley, NY, USA) were injected via intraperitoneal injection (i.p.). 60 mice were randomly assigned to three groups: 1. Sham group (n=20): mice were subjected to laminectomy and injected with PBS once daily for 2 weeks. 2. SCI vehicle group (n=20): mice were administrated with DMSO (i.p.) once daily for 2 weeks after SCI (n=20). 3. SCI+ISP group (n=20): mice received ISP (5mg/kg) (i.p.) once daily for 2 weeks after SCI. In primary spinal neuron, CSPG recombinant protein (5ug/ml), Chloroquine (CQ, 5ng/ml) or ISP (2.5 µM) were prepared for cell treatment. After drug incubated for 24 hours, cells were collected for detection.
Locomotion Recovery Assessment.
The (BMS) locomotor rating scale was widely used to assess the function recovery at 1D, 7D, 14D, and 28D after SCI. The BMS scores range from 0 of complete paralysis to 9 means normal locomotion. The mice (n=5 per group) were observed in an open field to account the BMS scores. The score was evaluated by two trained assistants blinded to the groups.
Spinal neuron culture
Neonatal C57BL mice within 1 day after born was collected. The spine tissues were cut and digested in trypsin EDTA for 5 min at 37 °C, and mixed with Dulbecco's Modified Eagle Medium (DMEM)/F12 to stop the digestion. After centrifugation at 1200 rpm for 5 min, cells were played on a disk coated with poly-D-lysine and cultured with DMEM/F12, 10% fatal bovine serum, 1% penicillin/streptomycin, and 2% B27 (Gibco, Grand Island, NY, USA). Cells were plated at a density of 3 × 104 cells/well in 100 mm culture. The medium was changed every other day.
Nissl Staining
10 um thickness sections (n=5 per group) were prepared for Nissl staining. In brief, sections were soaked in order with: 0.1% cresyl violet Nissl staining solution, 95% anhydrous alcohol, 100% anhydrous alcohol, then washed in xylene. The number of neurons were counted in randomly selected three sections.
Western Blot
The spinal cord tissues (1cm length from rostal to caudal of the lesion, n=5 per group) were collected and stored at −80°C for detection. After homogenized and centrifuged at 12,000 rpm for 30min, the supernatant was collected for protein assay. Proteins were run on 15% gel electrophoresis and transferred to PVDF menbrance. After 1% bovine serum albumin blocking for 2 hours, membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies all purchased from Cell signaling technology were as follows: anti-LC3B antibody (1:1000), anti-p62 antibody (1:1000), anti-LAMP2 antibody (1:1000), anti-STX17 antibody (1:1000), anti-synaptophysin antibody (1:1000), anti-PSD-95 antibody (1:1000), and anti-β-actin antibody (1:2000). After incubated with race matched HRP secondary antibodies for 1 h at 1:2000 dilution, the membranes were reacted with chemiluminescence (ECL) (Invitrogen) to visualize the protein.
Immunofluorescence Staining.
The lesion spinal tissues (n=5 per group) were stained with 4% paraformaldehyde and followed with 30% sucrose for several days. The sections were incubated with 0.1% Triton X-100 and goat serum for 2 hours and following primary antibody overnight at 4 °C. Primary antibodies all purchased from cell signaling technology were as follows: anti-STX17 antibody (1:200) and anti-LAMP2 antibody (1:100), anti-LC3B antibody (1:200), anti-NeuN antibody (1:400), anti-SYN antibody (1:100), anti-PSD-95 antibody (1:200), anti-GAP-43 antibody (1:200), anti-Neurofilament-L antibody (1:200). After three times washing with PBS, Alexa Fluor 488/568 secondary antibody (1:400) were incubated for 2 h. DAPI was used to stain nucleus for 5 minutes. And all images were captured with a Leica DMi8 microscope (Leica Micro-systems, Germany).
Reverse transcriptase-PCR (RT-PCR)
Total RNA (1ug) was isolated using a RNeasy plus Mini Kit (Qiagen). The PCR procedure was according to a SYBR Green PCR Kit (Qiagen). The target genes were normalized by β-actin. The primers are as following: SYN: 5’-CAGTTCCGGGTGGTCAAGG-3’ and 5’-ACTCTCCGTCTTGTTGGCAC-3’. PSD-95: 5’-GGCGGAGAGGAACTTGTCC-3’ and 5’-AGAATTGGCCTTGAGGGAGGA-3’. The results were determined via 2-△△Ct method.
Statistical analysis
Data were presented as Mean ± standard deviation (SD) and analyzed by SPSS 22.0. Two groups difference were analyzed using Student’s t-test and more than two groups via one-way ANOVA. P values less than 0.05 were considered statistically significant.