Animal breeding
32 male Sprague Dawley rats, aged 6~8 weeks, weighing 220-250 g, were housed in Laboratory Animal Centre of Nanjing Medical University in accordance to the animal experimental guidelines set by and with the approval of the National Institute of Health and the Nanjing Medical University.
SCI procedures
The rats were intraperitoneally injected with 1% pentobarbital sodium (50 mg/kg). The model of SCI was established as previously described[23]. Briefly, the spinous process and the vertebral lamina were removed to expose the spinal cord in a circular region at the T10 level. A graduated force of 70 g was placed over the exposed dura and left for 60 s to induce a compression injury. The rat models of SCI should meet the following characteristics to make sure all the models were in similar base level, including congestive edema of T10 segment, twitch of tail and hind limb, and neurogenic bladder.Each rat was feed separately, and the bladder of rats were given artificial massage twice a day until a reflex bladder emptying was established. Sham-operated mice received laminectomy without compression injury.
Basso-Beattie-Bresnahan (BBB) score
Lower limb motor function was evaluated using the BBB score, as previously described by Basso et al[24]. The rats were placed in an open field and two independent, blinded examiners observed these rats for 10 minutes individually. The rats were tested at the day5,10,15,20,25,30 post SCI surgery. In brief, the BBB scores of hindlimb motor function of SCI rats ranged from 0 (complete paralysis) to 21 (unimpaired locomotion), in which 0-7 mainly evaluated the movement of hindlimb joints, 8-13 mainly evaluated the gait and coordination of hindlimbs, and 14-21 mainly evaluated the fine movements of claws in the movement[19, 20].
Study design
The motor function of lower limbs in all rats was evaluated using BBB score after SCI operation at the day5,10,15,20,25 and 30, but the three rats with the highest BBB score (BBB high group) and the three with the lowest BBB score (BBB low group) were executed at the day10,20 and 30, respectively. Therefore,BBB high group always had three rats with the highest BBB score and BBB low group always had three rats with the lowest BBB score in every BBB score testing.The spinal cord tissues of the executed rats were removed and used for immunofluorescence detection in BBB high group and BBB low group at the day10,20 and 30, respectively.
Serum evaluation
Blood samples were taken from the eyeballs of all surviving rats for serum evaluation at the day5,10,15,20,25 and 30.Serum levels of interleukin (IL)-4, IL-10, major histocompatibility complex (MHC)-I and inducible nitric oxide synthase (iNOS) were detected using enzyme-linked immunosorbent assay (ELISA) according to manufacturer's instructions. The final concentrations of IL-4, IL-10, MHC-I and iNOS were interpolated from the determined standard curve of absorbance. After discovering that there may be some kind of pattern, we further compared serum IL-4 and IL-10 levels of the three rats with the highest BBB score (BBB high group) and the three with the lowest BBB score (BBB low group) at the day5,10,15 and 30, respectively.
Immunofluorescence
Spinal cord tissues were deparaffinized and rehydrated in coronal sections (4 μm thickness), then, the sections were blocked with 5% bovine serum albumin at room temperature for 30 min and then washed with PBS three times for 10 min. Sections were then incubated with corresponding primary antibodies overnight at 4°C, including F4/80, iNOS, Arginase(Arg)1 and NF200 (Invitrogen, Carlsbad, CA, USA), and incubated with secondary antibody (1:500; Abcam, Shanghai, China) for 3h. The slides were visualized using a fluorescence microscope (Olympus BX 51, Tokyo, Japan) and Image J (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) was used for quantitative analysis.
Statistical analysis
All data were presented as Mean± Standard Error of Mean (SEM), and were analyzed by two-way ANOVA repeated measurement using SPSS Statistics 20.0 (SPSS Inc., Chicago, IL, USA) for statistical significance. A P value < 0.05 was considered statistically significant.