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Page 4 of 13 Souza et al. Neuroimmunol Neuroinflammation 2019;6:12 I http://dx.doi.org/10.20517/2347-8659.2019.04

For ST36, the non-acupoint was located 5 mm lateral to the midline of the posterior face of the hindlimb,
[17]
and for GV3, approximately 1 cm lateral to the GV3 on the crest of the ilium .

Animals subjected to behavioral analysis received BV immediately after the SCI, and then weekly until
the fifth week; while rats subjected to methods of spinal tissue extraction (qPCR and Western blotting
analyzes) received BV only immediately after SCI. Spinal cord samples were collected at different time
points as described in the following topics.

Behavioral analyzes after SCI
For the evaluation of the locomotor capacity, the animals were submitted to the Basso, Beattie, and
[31]
Bresnahan (BBB) test as described previously . The BBB test, developed by Basso and colleagues, is a
well-established test widely used for investigating the mechanisms involved with the pathophysiology of
SCI and possible therapeutic targets. Each animal was placed individually in the center of the open field
and was observed for 4 min. During walking in the open field, locomotor parameters are observed and
compared with the BBB test scale which has scores ranging from 0 (no spontaneous movement in the hind
paws) to 21 (normal locomotion). The evaluation was performed weekly and sometimes up to twice in the
same week from the first to the thirty-fifth-day post-injury (1, 4, 7, 10, 14, 21, 28, 35 days after SCI) by two
raters blinded to the experiment. The values were represented as mean ± standard error.

[32]
Based on prior publications , a grid walk was constructed for rats using two parallel pieces of acrylic
[32]
plates (1 m in length) to hold metal bars (10 cm in length) with 1-4 cm apart . Before the injury, rats
were trained for 3 days on the apparatus. Each rat was allowed to cross the grid walk 3 consecutive times
at 35th day after injury and the number of “footfalls”, or the number of times that the animals’ hind
paws fall through the rungs were counted and represented as mean ± standard error. Animals unable to
[4]
move the hind limbs were assigned a maximum of 20 footfalls . The grid walk test evaluates the sensory-
motor coordination between hindlimbs and forelimbs and examines the deficits in descending motor
[32]
control . Although the BBB is a reliable test, the combined use of other tests as the grid walk can facilitate
[33]
the distinction of different motor and sensory impairments. Pajoohesh-Ganji et al. have shown that
combined scoring method can help the discrimination of different injury levels and produce less variability
than the individual tests, which can help to follow motor recovery after SCI.

Western Blotting
The Western blotting technique was used to evaluate IBA-1; BCL-2; NeuN e CNPase, where approximately
1 cm of the spinal cord at the lesion site was collected at different time points (days 1, 3, 5 and 7 after
the SCI). Initially, the collected tissue was mechanically macerated so that the cellular proteins were
fully lysed and homogenized in extraction buffer (Tris-HCl, pH 7.2) containing the protease inhibitor
cocktail (Protease Inhibitor Cocktail Tablets - Roche Diagnostics, Indianapolis, USA). Immediately after
extraction, the samples were centrifuged at 20,000 g for 40 min at 4 °C and the total protein concentration
of the supernatant was measured by the BCA method using a spectrophotometer (Thermo Scientific,
Washington, DC, USA). To perform the electrophoresis and transfer steps, 30 µg of protein per sample
were solubilized in a buffered solution [20% glycerol, 1 M Tris (pH 6.8), 4% sodium dodecyl sulphate
(SDS), 0.1 M dithiothreitol and 0.02% bromophenol blue; pH 6.8] submitted to SDS-polyacrylamide gel and
subsequent transfer of the protein to the nitrocellulose membrane. To promote blocking of non-specific
bonds, the membranes remained for 90 min in blocking solution [10% BSA dissolved in 1.5 M saline + 0.1%
Tween 20 Tris-HCl buffer (TBS-T)].

Overnight, the membranes remained incubated with the primary antibodies anti-β-actin (rabbit, dilution
1:3000, Abcam, Germany), anti-IBA-1 (goat, dilution 1:500, Santa Cruz, USA), anti-GFAP (mouse, dilution
1:1000, Abcam, Germany), anti-BCL-2 (mouse, dilution 1:500, Santa Cruz, USA), anti-NeuN (rabbit,