Design
A randomized, controlled, animal experiment.
Time and setting
The study was performed at the Nanfang Hospital Affiliated to Southern Medical University, China from April 2009 to June 2010.
Materials
Animals
A total of 54 adult Sprague-Dawley rats, of specific- pathogen free grade, weighing 250–300 g, male or female, were purchased from the Southern Medical University Animal Center (license No. SCXK (Yue) 20060015). Rats were housed at a constant temperature 23 ± 2°C and 12/12-hour light-dark cycles. The animal experiments were performed in accordance with the guidelines of Animal Ethics Committee of Nanfang Hospital.
Drugs
Methylprednisolone sodium succinate (No. H20080284) was purchased from Pharmacia & Upjohn, Belgium, Pfizer. Chemical structural formula of methylprednisolone sodium succinate is as follows:
Methods
Establishment of spinal cord injury models in rats
Rats were anesthetized by intraperitoneal injection of 15% chloral hydrate (3–4 mL/kg). Vertebrae T8–10 and their spinal meninges were exposed after carefully removing the lamina and pedicles. Steel weights were used to induce contusion models of spinal cord injury at T8–10 spinal segments, which were guided by a 10-cm long glass catheter according to an improved Allen’s method[26], following which the skin was sutured layer by layer. Rats in the control group only received surgery to expose the spinal cord without inducing any injury.
Drug intervention
The methylprednisolone group was slowly injected with methylprednisolone sodium succinate (30 mg/kg) for 15 minutes via the tail vein immediately after the model was established, which was performed three times over the following 24 hours. The spinal cord injury group and control group received an equal volume of physical saline (0.5 mL). Three days after the operation, all the animals were intraperitoneally injected with physical saline 10 mL/time, twice daily as well as sodium penicillin 400 000 U/day to maintain water and electrolyte balance and prevent infection. Rats in the spinal cord injury and methylprednisolone groups had their bladders squeezed twice a day to help release urine.
Behavioral examinations
Motor function was evaluated according to the Basso, Beattie, and Bresnahan scale scores[27-30]. The Basso, Beattie, and Bresnahan scale score (ranging from 0 to 21) represents the mobility of four limbs. Higher scores mean better motor function of limbs.
Histological examinations
After 3 days, rats were anesthetized with chloraldurat, following which they were perfused with 100 mL physical saline and 4% paraformaldehyde though a syringe needle inserted into the aorta (20 drips per minute for 3 hours). Then, a 3-cm incision was made around the back segments of the T8–10 layers and on each of the floor muscles. A 2-cm segment of the spine was collected from the injured segments by cutting both ends and a piece of complete spinal cord tissue was harvested after the lamina. The vertebral body and the surrounding scar tissue were carefully removed using a sclerectomy cutting device. After removal, the tissue was washed with 1 × Tris-buffered saline and placed in 4% paraformaldehyde. The spinal cord tissue was conventionally embedded in paraffin and cut into slices of 5 μm thickness for hematoxylin-eosin staining and immunohistochemistry analysis. For hematoxylin-eosin staining, eosin-methylene blue was used to differentiate the cytochylema and nucleus in the spinal cord tissue. Rabbit anti-rat Nogo-A antibody (1:400; Boster, Wuhan, China) was used as the primary antibody to mark the target protein at 4°C overnight. The samples were washed with PBS for 15 minutes and nonspecific antigens were blocked with 30% H2O2 for 5 minutes. Goat anti-rabbit IgG (1:200; Boster) was used as the secondary antibody and incubated at 37°C for 30 minutes. After incubation with diaminobenzidine kit (Boster), Nogo-A staining was seen as brown-yellow. The tissue was photographed using Olympus DP71 Image System (Olympus, Tokyo, Japan).
Nogo-A protein detection by western blot assay
After 3, 7 and 14 days, T8-10 spinal cord segments of the rats in all three groups were harvested and stored at –70°C. 40 mg tissue of each sample of spinal cord was grinded into cell lysate in 30 μL and homogenized in an ice bath. The supernatant was centrifuged at 7 500 r/min after boiling and mixed with an equal volume of 2 × sodium dodecyl sulfate sample buffer to obtain the total protein extract. The protein was transferred to polyvinylidene fluoride membrane (4°C, 2.5 hours, 50 V) after electropheresis on a 10% sodium dodecyl sulfate polyacrylamide gel. The membrane washed slowly with Tris-buffered saline solution three times for 5 minutes each, blocked in a solution of 1% bovine serum albumin and 0.02% Tween 20 in Tris-buffered saline, at 4°C for 6 hours. The membrane was then incubated with the primary antibodies, which were polyclonal rabbit anti-Nogo-A (1:400; Boster) and anti-β-actin antibodies, in blocking solution at 4°C overnight. Then the membrane was washed with Tris-buffered saline three times for 5 minutes, washed with 5 mL blocking solution, washed with Tris-buffered saline three times for 5 minutes, and incubated with goat anti-rabbit IgG (1:400; Boster) secondary antibody, followed by detection of the chemiluminescent signal (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Images were digitized and analyzed using a computerized image analysis software system (Image-Pro Plus6.0; Media cybernetics Inc., Silver Spring, MD, USA). The absorbance ratio of Nogo-A protein to standard protein (β-actin) represenst the relative levels of Nogo-A protein.
Statistical analysis
Data were expressed as mean ± SD. SPSS 13.0 (SPSS, Chicago, IL, USA) was used to analyze the data. Basso, Beattie, and Bresnahan scale scores and the expression of Nogo-A among the three groups were compared by one-way analysis of variance. One-way analysis of variance and least significant difference t-test (or Dunnett’s test) for multiple comparisons were adopted to have a separate analysis of effects. The results were considered statistically significant with a P level of 0.05.
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