Animals
A total of 120 specific pathogen-free (SPF) adult male Sprague-Dawley rats (Department of Animal Science, Peking University School of Medicine, Beijing, China) were used in this study. All rats were housed in separate cages under a 12-h light-dark cycle at 23 ± 1 °C and 50% relative humidity, and food and water were available ad libitum. All rats were acclimatized to the environment for at least 1 week prior to the experiment and were maintained as directed by the experimental animal care and use guidelines. The study was approved by the Animal Welfare Ethics Branch of the Peking University Bioethics Committee.
The animals were randomized into the following three groups (n = 40 in each group): (1) the sham+LV-pLent-U6-Puro group (the sham group); (2) the SCI+LV-pLent-U6-Puro group (the SCI group); and (3) the SCI+LV-SNCA-shRNA group (the SCI+knockdown [KD] group) (Fig. 1).
Construction of the lentiviral LV-SNCA-shRNA vector
Lentiviruses containing SNCA-shRNA (NM_019169.2) were constructed and synthesized by ShanDong ViGene Co., Ltd. (Shandong, China). The primers for SNCA were as follows: forward, 5′-GTGGCTGCTGCTGAGAAAAC-3′ and reverse, 5′-TCCATGAACGACTCCCTCCT-3′. The virus titre of LV-SNCA-shRNA was 1.0 × 10E9 TU/ml. In addition, an LV-GFP-SNCA-shRNA green fluorescent protein (GFP)-tagged lentivirus was constructed to verify the efficiency of transfection and knockdown.
Surgery and transfection
All rats received prophylactic antibiotic treatment with ampicillin sodium (80 mg/kg; Harbin Pharmaceutical Group Co., Ltd., Harbin, China) for 3 days before SCI surgery. The rats were intraperitoneally injected with 2% sodium pentobarbital (0.1 ml/kg) and placed in a prone position on the operating table. The limbs were fixed, and the upper chest was raised with a cotton pad. Along the T2 spine of each rat, the C8-T4 dorsal skin was dissected, the back muscle was peeled off layer by layer, and the T3 segment of the thoracic vertebra was dissected. The spinal cord was removed by performing a laminectomy of the T3 segment under a surgical microscope. In the sham group, the incision was closed layer by layer after the spinal cord was exposed, but no injury was induced. The SCI group was injured with a PSI-IH precision striking device (IH impactor; Precision Systems and Instrumentation, Lexington, KY, USA) after the spinal cord was exposed. The striking head was adjusted over the exposed T3 spinal cord segment, dropped so that it just touched the dural sac, and then raised by 2 cm. The force was set to 400 kilodynes, the compression time was set to 5 s, and the number of hits was set to one. The standards for successful generation of the rat SCI model were the presence of a contusion at the injury site, convulsions in both lower extremities, and spastic swaying of the tail. The force with which the spinal cords of the rats were impacted was monitored by a computer. Following SCI, 10 μl of lentivirus containing the target gene shRNA was injected in situ using a microsyringe. The sham group and the SCI group were given the same dose of an empty lentiviral vector. After injection, the dorsal tissue of each rat was sutured layer by layer. After the procedure, the rats were allowed to recover on a heated blanket and were placed in a clean cage for observation. Beginning immediately after surgery, the rats were injected subcutaneously with Ringer’s sodium lactate solution (5 ml) and ampicillin sodium twice daily (morning and afternoon) until the third day after injury. The bladders of the rats were squeezed 3 times daily after surgery until spontaneous urination was restored. All assessments and analyses were performed by experienced researchers who were unaware of the experimental design.
Behavioural tests
Hindlimb exercise score
Basso-Beattie-Bresnahan (BBB) motor function scores were used for evaluation of hindlimb motor function [13]. The rats were placed on a circular platform with a diameter of 2 m. The walking and limb activity scores of the hindlimbs were observed and recorded. In the first stage (0–7 points), the joint activity of the hindlimbs of the animals was scored. In the second stage (8–13 points), the gait and the coordination of the hindlimbs were scored. In the third stage (14–21 points), the fine movements of the claws were judged. The scores for the three stages together totalled 21 points. Each group was scored 1 day before surgery and 1, 3, 7, 10, 14, 21, and 28 days post-injury (dpi).
Footprint analysis
Gait behaviour and motor coordination were assessed on the 28th day after injury [14]. The forelimbs and hind paws were coated with dyes of different colours, and the animals were placed on a 7.5 cm × 100 cm runway covered with white paper. The animals were encouraged to walk straight to the finish line so that representative images of their gaits could be obtained and coordination could be assessed.
Tissue preparation
At the predetermined time points, each rat was anaesthetized, the diaphragm was cut, and the pericardium was opened. After blood was collected from the left apex, 5 mm of the spinal cord above and below the T3 injury site was quickly placed in a cryotube, frozen in liquid nitrogen, and stored in a − 80 °C low-temperature freezer. In addition, some rats were perfused with 150 ml of sterile 0.9% normal saline, and the right atrial appendage was cut at the same time. After the effluent liquid became clear, 300 ml of 4% paraformaldehyde (Biosharp, Beijing, China) was used for perfusion until the tissues of the rats became hard. The tissue near the injury site at the T3 level (5 mm above and below) was then removed and placed in paraformaldehyde overnight. The tissue was either dehydrated in xylene and gradient alcohol solutions, embedded in paraffin, and cut into 5-μm serial sections with a slicer or dehydrated in sucrose solutions (10%, 20%, and 30%), embedded in optimum cutting temperature (OCT) compound, cut into continuous 20-μm frozen sections with a microtome, and processed for immunofluorescence (IF).
Paraffin section histopathological staining
At 28 dpi, paraffin sections from each group were heated at 60 °C, placed in xylene I and II for 30 min, and placed in gradient alcohol solutions of 100%, 100%, 95%, 95%, and 80% for 5 min each. The sections were rinsed twice with steamed water.
- 1.
HE staining
A series of sections were stained with haematoxylin for 1 min, double-rinsed in distilled water, differentiated in 1% hydrochloric acid, double-rinsed in distilled water, and stained with eosin (Sigma-Aldrich) for 2 min.
- 2.
LFB staining
A series of sections were stained with a 0.1% Luxol Fast Blue (LFB; Sigma-Aldrich) solution, sealed for 8 h at 60 °C, washed with distilled water, and placed in 95% alcohol for 10 min. Each mixture was separated in a 0.05% lithium carbonate aqueous solution (Leagene, Beijing, China) for 10 s and in a 70% alcohol solution for 20 s. The above two steps were repeated until the grey and white matter (GM and WM, respectively) were clearly observable under the microscope.
The above sections were dehydrated by a conventional alcohol gradient (80%, 95%, 95%, 100%, 100% alcohol for 2 min), placed in xylene I and II for 10 min, and then sealed with neutral gum. Images of both haematoxylin-eosin (HE) staining and LFB staining were captured with a Nano Zoomer Digital Pathology system (Hamamatsu, Japan). Three blinded experimenters used Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) to calculate the staining density to quantify the myelin and lesion area. There were five animals per group. For each animal, five spinal cross-sections in the rostral-caudal plane taken from the level of the injury were analysed.
IHC
For immunohistochemistry (IHC), the 5-μm paraffin sections were dewaxed in water, and antigen retrieval was carried out in a pressure cooker with sodium citrate buffer (10 mM, pH 6.0; Boster Biological Technology, Ltd., Wuhan, China). The tissues were then blocked with 3% H2O2 to quench endogenous peroxidase activity. The tissues were blocked in 10% goat serum (Boster Biological Technology, Ltd) for 30 min, incubated with a rabbit anti-α-Syn antibody (1:2000; Abcam, Cambridge, MA, USA), rabbit anti-Iba1 (1:200; GeneTex, Inc., USA), and mouse anti-glial fibrillary acid protein (GFAP; 1:500; Santa Cruz, Dallas, TX) at 37 °C for 2 h, rinsed with PBS, and incubated with a goat anti-rabbit IgG secondary antibody (1:100; Zhongshan Jinqiao, Beijing, China) for 30 min. After washing, DAB (Zhongshan Jinqiao) was used for detection. The sections were then rinsed with water, the nuclei were counterstained with haematoxylin, the sections were placed in gradient alcohol solutions for 2 min each and in xylene I and II for 5 min each, and the slides were sealed with neutral resin. The sections were examined at × 20 magnification using an electron microscope (ECLIPSE 90i, Nikon, Japan) and Image-Pro Plus 6.0 to detect α-Syn staining in the GM and WM (through quantitative analysis of the mean optical density of the α-Syn+ cells). There were five animals per group. The average area of 4 different sections from each rat was calculated.
IF
The frozen sections were thawed at room temperature for 30 min. The sections were washed three times (for 10 min each) with 0.1 mmol/l PBS containing 0.1% Triton X-100 (Sigma-Aldrich) (PBS-TX). The sections were pre-incubated in permeabilization blocking buffer (0.1 mmol/l PBS, pH 7.3, containing 0.5% Triton) for 15 min at 37 °C. The sections were then blocked with 10% (v/v) goat serum (Boster Biological Technology, Ltd) for another 30 min and incubated overnight with a primary antibody at 4 °C. After washing with PBS-TX the next day (3 times for 10 min each), the sections were incubated with a secondary antibody for 1 h at room temperature and then washed with PBS-TX. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1 μg/ml; Sigma-Aldrich) for 5 min, and the sections were washed with PBS-TX and sealed with an anti-fluorescence quencher. Images were captured under a Leica DM4 B confocal fluorescence microscope (Leica Microsystems Inc., Wetzlar, Germany) with a Leica TCS SP8 system (Leica Microsystems Inc). Negative controls were incubated with the corresponding isotype serum instead of a primary antibody.
The following primary antibodies were used at the indicated dilutions: mouse anti-ionized calcium-binding adaptor molecule (Iba1; 1:100; Abcam, Cambridge, MA), rabbit anti-Iba1 (1:200; GeneTex, Inc., USA), mouse anti- glial fibrillary acid protein (GFAP; 1:500; Santa Cruz, Dallas, TX), rabbit anti-iNOS (inducible nitric oxide synthase, 1:400; Abcam, Cambridge, MA), rabbit anti-arginase-1 (Arg1; 1:400; GeneTex, Inc., USA), and rabbit anti-matrix metalloproteinase-9 (MMP-9; 1:100; Abcam, Cambridge, MA). The following fluorescent secondary antibodies were used at the indicated dilutions: Alexa Fluor 594-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:800; Jackson ImmunoResearch Laboratories, West Grove, PA), Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit IgG (H + L) (1:400; Jackson ImmunoResearch Laboratories), Cy3-conjugated goat anti-rabbit IgG (H + L) (1:200; Boster Biological Technology, Ltd), and Alexa Fluor 488-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:400; Jackson ImmunoResearch Laboratories).
There were 5 animals per group, and three sections from each animal at the same level (the injury level) were used to determine the number of positive cells or the mean optical density (mean optical density = integrated optical density (IOD)/area) for fluorescence quantification. The number of Iba1+ cells in each transverse section of the spinal cord was calculated by tissue fluorescence panoramic scanning at × 5 magnification. The remaining 4 random fields were observed at ×40 magnification to calculate the number of Iba1+/iNOS+ cells and the number of Iba1+/Arg1+ cells. The normalized mean fluorescence intensity of GFAP, Iba1, and MMP-9 in the central canal of the spinal cord was calculated. The experimenter who performed the analysis was unaware of the experimental groups of the rats.
RT-qPCR
For quantitative real-time PCR (RT-qPCR), total RNA was first extracted from spinal cord tissues with TRIzol (Invitrogen, Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s protocol. The ratios of absorbance at 260/280 nm and 260/230 nm were then determined using an ultraviolet-visible light spectrophotometer (NanoDrop 2000, Thermo, Waltham, MA, USA) to assess the purity and concentration of total RNA in each sample. A FastKing cDNA First Strand Synthesis Kit (Tiangen, Beijing, China) was used to synthesize cDNA from total RNA (2 μg/sample), and then RT-qPCR was carried out using SYBR Green PCR Master Mix (Tiangen, Beijing, China). The expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Three replicate wells were set up for each reaction. All primers used in this experiment were supplied by Sangon Biotech Co., Ltd. (Shanghai) (Table 1 lists the primer sequences). QuantStudio Design and Analysis Software (Applied Biosystems) was used to perform the following reaction: initial denaturation at 95 °C for 15 min and 40 cycles of 95 °C for 10 s, annealing at 55 °C for 30 s and 72 °C for 32 s (for SNCA, GAPDH, IL-1β, and CD86) or 40 cycles of 95 °C for 15 s, annealing at 60 °C for 35 s, and 72 °C for 32 s (for CD206 and IL-10). Melting curve analysis confirmed the primer specificity and determined the cycle threshold (CT) fluorescence values. The data were analysed by the 2−ΔΔCT method by researchers blinded to the experimental groups of the animals.
Table 1 Forward and reverse primer sequences for qRT-PCR Western blotting
Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (including protease inhibitor cocktail) [15]. After measuring the protein concentration using a BCA assay kit (Thermo Scientific, MA, USA), the protein was subjected to Western blotting. Equal amounts of protein from each sample were separated using 8%, 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at room temperature (22–25 °C) for 1 h and then incubated with a monoclonal rabbit anti-α-Syn antibody (1:10000, Abcam), a polyclonal mouse anti-α-Syn antibody (1:500, BD Biosciences), and a rabbit anti-α-Syn primary antibody (phospho-S129, 1:1000, Abcam) or a rabbit anti-β-actin antibody (1:10000; Abcam, Cambridge, UK) overnight at 4 °C. After incubation with the corresponding secondary antibodies for 1 h, the membranes were scanned using an Odyssey Sa imaging system (LI-COR Biosciences, Lincoln, NE, USA). The density of the results was quantified by two experimenters using ImageJ software (NIH, Bethesda, MD, USA), and the two experimenters were blinded to the characteristics of the samples studied.
ELISA
Serum samples were prepared by centrifugation using coagulant blood collection tubes. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the serum levels of TNF-α, IL-1β, IL-2, IL-10, IL-4, transforming growth factor-β (TGF-β) (Boster Biological Technology, Ltd., Wuhan, China), and IFN gamma (IFN-γ, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions on the 28th day after surgery. The absorbance was measured at 450 nm with a Multi-Mode Microplate Reader (Varioskan Flash, Thermo Scientific Inc., USA). The concentrations of TNF-α, IL-1β, IL-2, IL-10, IL-4, TGF-β, and IFN-γ were calculated from standard curves and are expressed in pg/ml.
Statistical analysis
The data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed in GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA). Comparisons between the two groups were performed using Student’s t test or the Mann-Whitney test, as appropriate. One-way and two-way analysis of variance (ANOVA) and Tukey’s test for multiple comparisons were used to analyse differences between groups. BBB motor function scores were analysed by two-way repeated-measures ANOVA followed by the Sidak multiple comparisons test. A p value < 0.05 was considered to indicate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.