Main Reagents and Instruments
The reverse transcription (RT)-PCR TERT assay kit was purchased from Roche (St. Louis, MO, USA). The GFAP detection kit, FITC-conjugated goat anti-rabbit antibody, mouse anti-GFAP antibody, rabbit anti-GFAP antibody, and neurofilament protein 200 (NF-200) were purchased from Sigma. The plasmid vector PBCA-NPS-TERT was provided by Invitrogen (Carlsbad, CA, USA). The PCR instrument was from BioRad Inc. (Berkeley, CA USA).
Spinal Cord Injury Model
A total of 120 male Sprague–Dawley rats (clean grade, 180–220 g) were purchased from the Experimental Animal Center, Xinjiang Medical University (license No. SYXK (Xin) 2003-001). The rats were randomized into SCI only group, TERT siRNA group, and sham group (n = 40 rats per group). All rats were anesthetized with a combination of 2 mL ketamine, 1 mL atropine, 2 mL diazepam, and 5 mL 0.85 % sodium chloride via intraperitoneal injection (0.5 mL/100 g). After anesthesia, the modified Allen’s weight drop method was used to induce spinal cord injury at the T8 segment in the TERT siRNA and SCI only groups under aseptic conditions [11]. After modeling, the bleeding was stopped and the incision sutured, and the bladder was manually squeezed every 8 h to help urination until spontaneous voiding. In the sham group, the spinal cord was exposed but not damaged. On the basis of GenBank bioinformatics analysis, we designed sense and antisense nucleotides targeting astrocyte TERT mRNA in rats (antisense oligodeoxynucleotide 5′-GTTAGGGTTAG-3, sense oligodeoxynucleotide 5′-CTAACCCTAAC-3′. In this study, we used PBCA-NPs-TERT to effectively inhibit TERT expression. In the TERT siRNA group, PBCA-NPs-TERT saline solution (91.861692 pmol/μL) was injected at a dose of 50 μg/kg body mass into the surgical site at 30 min after modeling, twice per day. No administration was performed in the SCI only groups and sham groups.
Sample Collection
In each group, five rats were sacrificed under anesthesia at days 1, 3, 5, 7, 14, 28, 42, and 56. Under sterile conditions, 50 mg of spinal cord tissue, 5 mm in length, was taken from the central zone of the injured spinal cord, and was then rinsed with 0.1 % DEPC water and stored in nuclease-free cryovials at −80 °C.
Pathological Observation of Spinal Cord Glial Scar
Hematoxylin-Eosin Staining
Two sections from rats were taken at each time point for hematoxylin-eosin staining to observe the morphology of spinal cord tissues, changes in glia and nerve cells, and glial scar hyperplasia.
Immunofluorescence Detection of GFAP and NF-200 Expression
Frozen sections were rinsed with 3× PBS (once for 10 min), blocked with 10 % goat serum for 1 h at room temperature, and incubated at 4 °C overnight. Sections were rinsed with 3× PBS (once for 10 min), and incubated in the following primary antibodies: chicken anti-GFAP/NF-200 polyclonal antibody (1:2,000), mouse anti-CDllb/c monoclonal antibody (1:200), and rabbit anti-Fibronectin polyclonal antibody (1:200), for 24 h at 4 °C. Sections were then rinsed with 3× PBS (pH 7.4; once for 5 min), followed by incubation with secondary antibodies (DyLight488 green fluorescence-labeled goat anti-mouse IgG; Texas Red-labeled rabbit anti-chicken IgY) for 1 h in the dark at 37 °C. After 3× PBS rinses (once for 5 min), the sections were observed under a 200× fluorescence microscope. Numbers of GFAP and NF-200-positive cells in five fields of view were counted and the area of positive cells was calculated using Image-ProPlus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA).
Western Blotting Assay for Detection of TERT and GFAP Expression
Total protein extract from approximately 50 mg of spinal cord scar tissue was prepared from the various time points. Protein samples (20 μL) from each group were resolved in a 7.5 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto film. The samples were incubated in rabbit anti-rat TERT monoclonal antibody (1:100) for 2 h at 37 °C, and then stored at 4 °C overnight. After incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (1:50) for 1 h at room temperature, the samples were colored with chromogenic substrate for 2 min in the dark. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control, and a gel imaging analysis system was employed for semi-quantitative analysis. The ratio of telomerase to GAPDH was calculated.
Detection of TERT and GFAP mRNA by RT-PCR
Total RNA was extracted using the Trizol method according to the manufacturer’s instructions (Invitrogen). Primers sequences used were: TERT: upstream, 5′-TCCGCACGTT GGTTGCCCAG-3′, downstream, 5′-CCTCTCACCGCGCTCGCA AA-3′ (product size = 203 bp); GADPH: upstream, 5′-GCTCTCTGCTCCTCCCTGTTCT-3′, downstream, 5′-CAGGCGTCCG ATACGGCCAAA-3′ (product size = 450 bp); GFAP: upstream, 5′-CTG AATTC TGCTGGCTTCAAGG-3′, downstream, 5′-CTAAGCTTGCTCTGCGTTGCGG-3′ (product size = 624 bp); and β-actin: upstream, 5′-GCGGGAAATCGTGCTGA CATT-3′, downstream, 5′-GATGGAGTTGA AGGTAGTTTCGTG-3′ (product size = 314 bp). PCR was performed as follows: denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles, followed by extension at 72 °C for 5 min. PCR products were analyzed on a 15 g/L agarose gel. TERT and GFAP mRNA expressions were confirmed by comparing the PCR band intensity between TERT and GAPDH as well as between GFAP and β-actin. All gene expression data were calculated as 2−△△Ct.
Statistical Analysis
Data are expressed as mean ± SD, and were analyzed using SPSS 19.0 (IBM, Armonk, NY, USA). Analysis of variance for a completely randomized design was used to compare differences between the three groups. A least significant difference test was used for comparison between groups. P < 0.05 was considered statistically significant.