Surgical Procedure
Adult (2–3 months old) female C57BL/6 Wild Type (WT) mice and homologous ApoE-/- mice weighing 18 - 20 g were used in this experiment. Animals were obtained from the Experimental Animal Center of Chongqing Medical University and housed in the standard cage in the Specific Pathogen Free (SPF) grade room with a 12-hour light/dark cycle, room temperature 22 - 24℃, relative humidity 65% - 75% and free access to food and water. All experiments were carried out in strict accordance with Guiding Opinions on the Treatment of Experimental Animals promulgated by the Ministry of Science and Technology of the People’s Republic of China in 2006. Disposal of animals throughout this experiment complied with the standards of the Ethics Committee of Chongqing Medical University.
WT mice and ApoE-/- mice were anesthetized by intraperitoneally injecting 1% sodium pentobarbital (80 mg/kg, i.m.). Throughout the surgery, the mice kept deep sedation. A sterile and limited laminectomy was performed at the first lumbar vertebra without disrupting the dura. The exposed dorsal surface of the cord was subjected to clamping injury with a symptom of a spasmodic tail and tremulous hindlimbs by a serrefine for 25s [51]. Heating facility was supplied to mice until they fully recovered from the anesthesia. After surgery, muscles and skins were sutured in layers. Penicillin (40,000 U, i.m.) had been applied for 3 days to prevent infection. Abdominal massage was implemented twice daily to improve bladder dysfunction until mice resumed spontaneous micturition.
All the mice subjected to same degree injury were selected into the study. WT mice were divided into: sham group, control group, EA group, EA+COG112 (ApoE mimetic peptides administration) group. ApoE-/- mice were divided into: EA group, EA+COG112 group, with 18 mice in each group. In addition to postoperative basic care, different groups of mice received the following treatment: the sham group underwent only laminectomy on L1 without damaging the spinal cord; the control group received no treatment but free access to food and water; the EA group received EA treatment; the EA+COG112 group received the combination of EA treatment and COG112 treatment.
EA Treatment
In the EA and EA+COG112 groups, mice received EA treatment. EA stimulation was performed at the bilateral “Zusanli” (ST 36, located at the anterior aspect of the hindlimb, straightly 2 mm below the knee joint) and “Sanyinjiao” (SP 6, located at the posterior to the tibia, 3 mm above the medial malleolus). Stainless steel needles of 0.25 mm × 13 mm were inserted to a depth of 3-5 mm at each acupoint. Then, the needle handles were linked to the output terminals of an electronic acupuncture instrument (SDZ - II, Suzhou Medical Products Co., Ltd., China). An intermittent wave was used to stimulate acupoints, and the hind limbs showed a mild twitch. The intensity lasted 10 min every time for each. EA had been administered for 4 weeks (six days for continuous treatment followed by one day off per week), starting from the first day post-surgery.
COG112 preparation and application
COG112, one of ApoE mimetic peptides with anti-inflammation character used in this experiment, was synthesized by the laboratory (Gill Biochemical Co., Ltd., Shanghai, China). The peptide sequence of COG112 is acetyl-RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-amide. All peptides were purified by high-performance liquid chromatography (HPLC) with a purity of >95%.
In the COG112 and EA+COG112 groups, mice received COG112 treatment. COG112 was formulated with lactated ringer's buffer and the single dose of each mouse was 1 mg/kg, twice daily for 4 weeks. The first injection was performed at 3 h after surgery by intraperitoneal injection.
Behavior test
Basso Mouse Scale (BMS), an effective scoring method for injured animals to assess the hind limb locomotor function, ranges from 0 to 9, in which 0 denotes complete paralysis and 9 denotes normal movements. Two raters who were familiar to the scoring criteria and blinded to this experiment scored the hind limb locomotor function of mice according to the ankle joint mobility, coordination, paw status, trunk stability and tail posture. Each mouse was observed for 5 min in an open field, and the average of two raters’scores was taken as the final score. This test was performed on day 1, 3, 7, 14 and 28 after SCI, and only mice scored 0 - 0.5 were applied to this experiment.
Tissue Preparation
On the 28th after SCI, mice were deeply anesthetized and transcardially perfused with 0.01M phosphate buffered saline (PBS), followed by 4% paraformaldehyde solution (4% in 0.01 PBS) at room temperature. After fixated with the universal tissue fixative (Google Biotech Co., Ltd., Wuhan, China) for 24 h, the tissue was completely dehydrated with graded ethanol, permeabilized with xylene, embedded in paraffin and cut into 6 μm thick sections for histological analysis.
Hematoxylin/eosin (HE) staining
Tissue sections were deparaffinized with xyleneⅠandⅡ, and immersed in hematoxylin solution for 3 min, following by rinsing quickly in distilled water. Then the slides were differentiated in HCL/95% alcohol (1:50) solution for 10 s. After washing in distilled water for 5 min, the sections were stained by 0.5% eosin for 10 s, dehydrated by gradient ethanol for 3 min (95%, 100%), and then transparentized by xylene for 1 min. The sections were examined under a fluorescent microscope (BX 53, Olympus, Japan). The pathological scores were determined by edema, hemorrhage, tissue cavity, neuronal apoptosis or necrosis, and inflammatory cells infiltration and represented the degree of SCI over a range from 0 to 4 points: 0 for none or minor, 1 for limited, 2 for intermediate, 3 for prominent, and 4 for widespread. Two raters who were familiar to the scoring criteria and blinded to this experiment scored pathological changes.
Immunofluorescence (IF)
For immunofluorescence staining, Tissue sections were deparaffinized by xyleneⅠandⅡ,rinsed, blocked with goat serum (Boster, China) at 37 °C in a humidified atmosphere for 1 h, and incubated with primary antibodies, including rabbit anti-ApoE (1:400, Abcam, America), rabbit anti-Nrf2 (1: 400, Abcam, America) overnight at 4 °C. The sections were rinsed again with PBS and incubated with secondary antibodies (goat IgG conjugated with Alex 492, 1:200, Earthox, Shanghai) for 1 h at 37 °C in a humidified atmosphere in the dark. Nuclear dye (4′,6-diamidino-2-phenylindole, Beyotime Bio, C1005, China) was applied to sections for 5 min. Then, the sections were rinsed again and mounted with an antifade mounting medium (Beyotime Bio, China). The samples were observed under a fluorescence microscope.
Transmission Electron Microscopy (TEM)
Mice were deeply anaesthetized and transcardially perfused with PBS followed by a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde (1:1). The spinal cord containing the lesion epicenter (5 mm) was removed and fixed for overnight at 4℃. Samples were placed in 1% osmium tetroxide and dehydrated in ascending graded ethanol and acetone. Following embedding in Epon-Araldite resin, tissues were cut into ultrathin sections using a reichert ultramicrotome and then collected with copper grids. After staining with uranyl acetate and lead citrate, the sections were photographed by a Philips EM420 electron microscope. Image Pro Plus software was used to quantify the ratio of demyelinated axons to total axons.
Western blot (WB) analysis
On the 28th after SCI, mice were deeply anaesthetized and transcardially perfused with 0.01 M PBS. Spinal segments (1cm) containing the lesion were immediately removed and grounded on ice in a mixture of RIPA lysis buffer (Beyotime Institude of Biotechnology, China) containing 1% protease inhibitor. The homogenate was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was diluted with 5× protein loading buffer and then heated at 95°C for 5 min. Proteins in samples containing the same mass of protein (40 μg) were separated by the 10% SDS polyacrylamide gel electrophoresis system, and then transferred to PVDF membrane. After being blocked the non-specific binding site with 5% skim milk for 2 h at room temperature, the membranes were incubated with the following antibodies at 4 °C for overnight: rabbit anti-β-actin (1:5000, CST, USA) ), rabbit anti-Nrf2 (1: 1000, Abcam, America), rabbit anti-HO-1 (1:2000, Abcam, America), rabbit anti-NQO1 (1:10000, Abcam, America), rabbit anti-ApoE (1:1000, Abcam, America). Horseradish peroxidase-labeled goat anti-rabbit IgG (1:100; Boster) was then incubated for 1 h at 37 °C. Protein bands were exposed for 1 minute with Enhanced Chemiluminescence (ECL) detection reagent (Applygen Technologies Inc., Beijing, China). The intensity of all protein bands was measured by Image J Software and the relative band intensities of the target proteins were normalized to those of the respective β-actin internal controls. All western blot experiments were performed in triplicate.
Quantitative reverse transcription polymerase chain reaction (qRT- PCR)
The mRNA expression levels of pro-and anti-inflammatory cytokines were determined at 28 dpi. Mice were transcardially perfused with 0.01M PBS as mentioned earlier. Standardized areas of spinal cord tissue (5 mm cephalad and 5 mm caudal to the lesion center) were harvested. Total mRNA was extracted using the RNAiso Plus (TaKaRa, Japan), according to the manufacturer’s instructions. Reverse transcription to cDNA was performed following the reaction protocol provided with the PrimeScript® RT reagent Kit With gDNA Eraser. Quantitative PCR was conducted by the CFX 96 real-time PCR detection system (Bio Rad, America) under universal cycling conditions (30 s at 95℃ followed by 40 cycles of 5 s at 95°C, and 30 s at 60°C). The reaction mixture contained fast SYBR Green master mix (Applied TaKaRa), 10mM forward and reverse primers (TaKaRa), RNase free water, and cDNA in a total reaction volume of 10 µl. Relative quantification of gene expression was accomplished by using the 2−ΔΔCT method and data were normalized to the most stable reference genes. The primer sequences used are shown in Table 1.
Table 1 Forward and reverse primer sequences for qRT-PCR
Gene Name
|
Forward primer (5′-3′)
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Reverse primer (5′-3′)
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TNF-α
|
FP: 5′-GCCAACGGCATGGATCTCAA-3′
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RP: 5′-TGACGGCAGAGAGGAGGTTGA-3′
|
IL-6
|
FP: 5′- CTTGGGACTGATGCTGGTGAC-3′
|
RP: 5′-TTCTCATTTCCACGATTTCCCA-3′
|
IL-1β
|
FP: 5′-GCTGCTTCCAAACCTTTGACC-3′
|
RP: 5′-AATGAGTGATACTGCCTGCCTGA-3′
|
IL-10
|
FP: 5′- TTGCCAAGCCTTATCGGAAAT-3′
|
RP: 5′-TGAGGGTCTTCAGCTTCTCACC-3′
|
TGF-β1
|
FP: 5′-GAGGCGGTGCTCGCTTTGTA-3′
|
RP: 5′-CGTTGTTGCGGTCCACCATTA-3′
|
β-actin
|
FP: 5′-AGATTACTGCTCTGGCTCCTAGC-3′
|
RP: 5′-ACTCATCGTACTCCTGCTTGCT-3′
|
Statistics
All statistical analyses were performed using GraphPad Prism 5.01 software (GraphPad Software, Inc., San Diego, USA), in which data were expressed as Mean±standard error of the mean (SEM) and analyzed through ANOVA. P < 0.05 denoted statistical significance. Two-way ANOVA was applied to BMS scores. One-way ANOVA and Bonferroni's multiple comparison test were used to analyse differences among groups. At 95% confidence interval, differences were considered statistically significant when P<0.05.