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Souza et al. Neuroimmunol Neuroinflammation 2019;6:12 I http://dx.doi.org/10.20517/2347-8659.2019.04 Page 5 of 13

dilution 1:1000, Millipore, USA), anti-CNPase (mouse, dilution 1:750, Millipore, USA) at 4 °C in a solution
containing TBS-T and 5% BSA. After washing they remained incubated with secondary antibodies
peroxidase-conjugated (Abcam, Germany) anti-rabbit (dilution 1:5000), or anti-goat (dilution 1:2000), or
anti-mouse (dilution 1:1000) diluted in a solution containing 5% BSA in TBS-T for 1 h.

For detection of the bands, the membrane was incubated with chemiluminescence reagents (ECL; Bio-
Rad, Hercules, CA, USA) and suffered 90 s of exposure in ChemiDoc XRS + Imaging System (Bio-Rad,
Hercules, CA, USA). β-actin was applied in the same blot technique for densitometric measurements to
normalize the intensities of specific bands using the ImageLab program of Bio-Rad (Bio-Rad, Hercules,
CA, USA).

Real-time PCR
Spinal cord samples from animals of all groups and collected at different time points (days 1, 3, 5 and 7
after the SCI) were submitted to total RNA extraction using QIAzol Lysis Reagent (Qiagen) and eluted
in 30 µL of RNAse free water. For mRNA analyzes, High Capacity cDNA Reverse Transcription kit
(Thermo Fisher Scientific) was used to prepare cDNA in a final concentration of 50 ng/µL, following
the manufacturer’s protocol. For qRT-PCR, the following reaction was prepared: 5.9 µL of nuclease-free
water, 7.5 µL of Power SYBR® Green PCR Master Mix, 10 µM of forward and reverse primers and 5ng of
diluted cDNA. Quantitative gene expression was normalized to the expression levels of housekeeping gene
GAPDH. The target genes from the experimental group were compared with those from the control group
using the 2-ΔΔCT method. Primer sequences for each of the mRNA targets were the following: Arginase-1
forward: 5’ ATATCTGCCAAGGACATCGTG 3’, reverse: 5’ AGGTCTCTTCCATCACTTTGC 3’; iNOS
forward: 5’ GGAGCAGGTTGAGGATTACTTC 3’, reverse: 5’ TCAGAGTCTTGTGCCTTTGG 3’; TGF-β
forward: 5’ TGGCGTTACCTTGGTAACC 3’, reverse: 5’ GGTGTTGAGCCCTTTCCAG 3’; COX-2 forward:
5’ TCAAGGGAGTCTGGAACATTG 3’, reverse 5’ GCTTCCCAACTTTTGTAACCG 3’; GAPDH forward:
5’ CCATCAACGACCCCTTCATT 3’, reverse: 5’ GACCAGCTTCCCATTCTCAG 3’.

Statistical analysis
All statistical analyses and construction of the graphs were performed by GraphPad Prism 5.0 software
(San Diego, CA, USA).To perform the BBB test analysis, the data were submitted to a two-way analysis of
variance (ANOVA) for repeated measures followed by Bonferroni post-test. For the analysis of the Grid
Walk test, Western blot and qRT-PCR, the data were submitted to one-way ANOVA followed by Bonferroni
post-test. For data that did not present normal distribution, the Kruskal Wallis test was performed followed
by Dunn’s post-test. All dates from this experimental protocol were expressed as mean ± standard error of
the mean. The statistics were considered significant only when P < 0.05.

RESULTS
Apipuncture promotes functional recovery in SCI rats
After SCI, rats show 0-1 scores in the BBB test, meaning no spontaneous movements in the hind limbs.
Compared to CTL-SCI and BV (NP)-SCI groups, BV (ST36 + GV3)-SCI groups showed significant higher
scores in BBB test, at 7, 14, 21 and 35 days after SCI (Two way Anova for repeated measures, P < 0.001). In
the same way, the BV (ST36 + GV3)-SCI group had a significantly lower footfalls in the grid-walking test in
comparison with the CTL-SCI and BV (NP)-SCI groups (One way ANOVA followed by Bonferroni test; P
< 0.001) [Figure 1].

Apipuncture changes polarization phenotypes of M1 (iNOS) and M2 (Arg-1 and TGF-β)
BV (ST36 + GV3)-SCI group showed significant lower expression of iNOS mRNA (M1 marker) than CTL-
SCI and BV (NP)-SCI groups in the 3rd and 5th day after SCI (One way ANOVA followed by Bonferroni