Animals and groupings
All experimental procedures were performed in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Institute of Translational Neuroscience Center of Sichuan University, Chengdu, China and by the institutional ethics and licensing committee of Chengdu Medical College, Chengdu, China.
A total of 144 female Sprague-Dawley (SD) rats (each weighing 200±20 g) were purchased from the Animal Experimental Center, Medical Sciences Department of Sichuan University. The animals were individually housed, in quiet room, under 12/12 hours light/dark condition with free access to water and food. Animals were randomly divided into four groups: sham; spinal cord contusion injury (SCC); vector; and lentivirus mediated siRNA interference (IL-1β-RNAi-LV). Spinal cord contusion injury group was further divided into the following post-injury subgroups with six animals each: 6 hours (6h), 12 hours (12h,) 1 day (1d), 3 days (3d), and 28 days (28d). This one and lentivirus-mediated siRNA interference groups consisted of the following subgroups of six animals each: 3 days (3d), 7 days (7d), and 28 days (28d).
Preparation of lentiviral vector
Information on siRNA fragment with the highest interference efficiency was provided by Gene-Copoeia, Guangzhou, China. We then constructed the IL-1β lentiviral expression vector, which expresses a gene encoding with red fluorescent protein (RFP). Thereafter, IL-1β-lentiviral expression vectors (5 μg) and viral packaging vectors (1 μL) were co-transfected into packaging cell line (293T) to produce lentiviral particles (IL-1β-RNAi-LV). The viral supernatant was harvested at 48 hours post-transfection and filtered through a 0.45 μm cellulose acetate filter. Then 5 mL of cell supernatant containing lentivirus was centrifuged (3500 × g, 25 minutes). The precipitate was re-dissolved in 500 μL phosphate-buffered saline. Finally, the lentivirus was frozen at 80°C. The negative plasmid was also packaged and used as a negative control, designated as NC-LV, which theoretically had no effect on any gene.
Spinal cord contusion animal model
Rats were anesthetized using intraperitoneal (IP) injection of 3.6% chloral hydrate (1 mL/100 g). Assess the depth of anesthesia by judging the toes of rats 。Under aseptic conditions, an incision was made along the dorsal midline to expose the soft tissues and muscles, the yellow ligaments overlying the spinal end plate at T11, and finally the spinal cord. A micro-syringe with 10 μL virus mixture was injected 4 mm deep, 5μl each caudally and cranially to the spinal cord. At 48 hours post injection, bilateral laminectomy was performed at T11. To induce SCC, a 10 g weight was dropped from a height of 30 mm onto the exposed cord at T11. Urinary bladders of rats were manually expressed twice a day for one week until normal urination was restored. Rats in the sham group received laminectomy without cord injury. The animals were placed in a warm environment (33-35°C) 24 hours following the procedures. During this period, they were observed for infection (blood in urine, foul odor, whitish color).
Locomotor function assessment
Restoration of hind limb function was assessed by the Basso Beattie Bresnahan (BBB)  locomotor scale on days 1, 3, 5, 7, 14, and 28 post-injury. The 28-day group animals were placed in a 2 meter square cardboard box and observed by three people who were blinded to treatment of the rats. The animals were observed according to the standard BBB grading standards and the recovery of motor function in the hind limbs was recorded. All observations were performed simultaneously. Scoring criteria were as follows: 0-7, joint activity; 8-13, gait and coordination function; 14-21, claw movement. Maximum score was 21 and hind limb paralysis was scored as 0.
GeneMANIA was used (www.genemania.org; University of Toronto, Toronto, Canada) to predict the relationships and functions of IL-1β and other genes of the rat species.
Quantitative polymerase chain reaction analysis (qPCR)
Rats were anesthetized by IP injection of 3.6% chloral hydrate (1 ml/100 g) and perfused with 4% paraformaldehyde solution for 30 minutes. Damaged spinal cord tissues were carefully dissected and pre-treated with the total RNA isolation reagent superfecTRI (Shanghai Pufei Biotechnology, Shanghai, China) and then centrifuged at 33500g for 10 minutes before homogenizing. Total mRNA was extracted per the manufacturer’s protocol and reverse transcription to cDNA was performed with the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). The kit was used for amplification, which included 160 μL TE, 40 μL cDNA, 12.5 μL 2X PCR Mix, 0.6 μL forward primer, 0.6 μL reverse primer, 0.6 μL TaqMan probe, and 7.7 μL water. The PCR mixture underwent 45 cycles of 95°C for 2 minutes, 95°C for 15 seconds, 52°C for 20 seconds, and 60°C for 40 seconds to gain PCR amplification products. During the process, the ΔCT value was recorded, the relative content based on 2−ΔΔCT value was calculated and inspected. Relative expressions were calculated with normalization to β-actin values. Design and synthesis of primers (Table 1) and fluorescent probes were made. Gene sequences were obtained from GenBank (www.ncbi.nlm.nih.gov/gene; National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, MD, USA) and primers were designed using Primer Premier 5 (Premier Biosoft International, Palo Alto, CA, USA). TaqMan fluorescent probes were constructed based on Applied Biosystems 7300 Real Time Quantitative PCR System (Thermo Fisher Scientific) filter wavelength, FAM was selected as the fluorescent reporting group and TAMRA was chosen for the quenching group. The primers and probes were synthesized by Sangon Biotech, (Shanghai, China).
Western blotting was used to determine the changes in IL-1β protein in the spinal cord in response to different treatments. The spinal cords at the lesion sites were harvested at different time points post-injury. The rats were deeply anesthetized with IP injection of 3.6% chloral hydrate (1 mL/100 g), and the spinal cords were dissected immediately. The tissues were homogenized on ice in 400 μL of RIPA buffer (Thermo Fisher), containing a cocktail of phosphatase and protease, then centrifuged at 12000 × g for 30 minutes. Protein concentration of each sample was assayed with BCA reagent (Sigma, St. Louis, MO, USA). A 20 μL aliquot of the samples was loaded and electrophoresed on 8% and 12% SDS polyacrylamide gel for 1.5 hours at 120 volts. Proteins were transferred from the gel to a polyvinylidene difluoride membrane. Then the membrane was blocked with 5% nonfat dry milk for 120 minutes. Primary antibody of β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-IL-1β (1:100, rabbit; Abcam, Cambridge, MA, USA) were separately incubated with two target bands overnight at 4°C after the membranes were rinsed thrice in phosphate-buffered saline containing 0.05% Tween-20. The membrane was incubated for 2 hours with HRP-conjugated goat anti-rabbit IgG (1:2,000; Santa Cruz) or goat anti-rabbit IgG (1:5,000; ZSGB-BIO, Beijing, China) for 2 hours at room temperature. Finally, the membranes were rinsed thrice in buffer and the immune complexes were quantified using the Alpha Imager 2000 (Alpha Innotech, San Leandro, CA, USA) with Western Lightning Plus enhanced chemiluminescence substrate (Perkin Elmer, Waltham, MA, USA). Protein densitometry analysis was performed by Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA, USA).
Rabbit IL-1β polyclonal antibody (1:100; Abcam) was used as the primary antibody, while goat anti-rabbit IgG (1:100; Jackson Immunoresearch, Westgrove, PA, USA) was used as the second antibody, and DAB chromogenic liquid was applied for detection of positive expression. The epicenters of injured cord tissues were embedded in methyl methacrylate plastic after serial dehydration with a graded ethanol series to xylene. The immune-positive cells were identified using an optical microscope. Five immunohistochemical slices were taken from each tissue. The ventral and dorsal horns of the spinal cord could be separated based on the vertical straight line drawn between central tube and dorsal medial sulcus of the spinal cord. Immune-positive cells with nuclei in the ventral horn were counted under a 10×-40× stereo microscope (Motic, Carlsbad, CA, USA).
Primary antibodies used in this study contained rabbit IL-1β polyclonal antibody (1:100; Abcam), rabbit AKT1 polyclonal antibody (1:150; Abcam), and NeuN (1:50, ZSGB-BIO). Damaged spinal cord tissues were dehydrated, fixed, and embedded in paraffin, then cut into 5 µm thick slices. The epicenter of injured spinal cord tissues were incubated in blocking buffer (5% goat serum and 0.3% Triton X-100 in phosphate buffer saline) for 30 minutes at 37°C before incubating overnight with the primary antibodies at 4°C. Secondary antibodies were incubated at 37°C for 1 hour before washing with the phosphate buffer saline. The nucleus was stained using DAPI (0.8 μg/mL, Beyotime, Shanghai, China). The tissues were then mounted onto slides and viewed under a Leica fluorescence inverted microscope (Leica Microsystems, Wetzlar, Germany).
Data were presented as mean ± standard deviation. Statistical analysis of physiologic parameters was performed with independent sample T test and one-way analysis of variance (ANOVA). The Dunnett family error rates were chosen for unequal sample sizes, otherwise the LSD test was selected. Statistical analysis was performed using SPSS17.0 software (IBM, Armonk, NY, USA). Statistical significance was defined as a P-value of < .05.