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iNOS, IL-1β, TNF-α; whereas M2 presents anti-inflammatory profile by promoting the release of anti-
inflammatory cytokines such as IL-10, phagocyte myelin remnants and inhibiting factors to tissue
regeneration [18,23,25,27,28] . After SCI, there seems to be a dominance of the M1 phenotype [25,27] , and although
more recent studies have shown that microglia can have multiple activation phenotypes (a “full spectrum
[22]
of activation”) and consider the model of two dualistic microglial state is too simplistic for revision ;
therapeutic approaches that stimulate greater phenotypic polarization of microglia/macrophages in the M2
profile are likely to represent a promising tool [18,23,25] .

Thus, in order to expand the previous results, using the same SCI model and followed the same therapeutic
methodology, the present study aims to evaluate whether BV injection at ST36 and GV3 acupoints could
reduce neuroinflammation by modulating the microglia/macrophages polarization in the M1 and M2
status, reduce apoptosis and promote the neuroprotection of neurons and oligodendrocytes in the SCI
model by compression in rats.

METHODS
Spinal injury compression and groups
Adult male Wistar rats, weighing between 270 g and 300 g, were kept in 12/12-h light and dark cycles
at a constant temperature, with food and water ad libitum. All procedures were approved by the Ethics
Committee on Research of the Federal Rural University of Rio de Janeiro (23083.005880/2013).

Before the surgical procedure, the rats were anesthetized with a mixture of ketamine and xylazine (200
and 10 mg/kg, i.p.; respectively, FortDodge, São Paulo, Brazil). SCI induced by compression was similar to
[17]
[29]
previously described by Vanický et al. . As previously described , after exposure of the T10-L1 vertebra,
a 2-French Fogarty catheter (Edwards Lifesciences, EUA, CA, USA) was inserted into the epidural space
through a small hole in the vertebral arch (mini-laminectomy) of T10 and advanced cranially until the
center of the balloon rested at the T8 and T9 level. The balloon catheter was inflated with 15 μL of saline for
5 min using a Hamilton syringe (type 1705). A sham-operated group was submitted to a mini-laminectomy
[17]
without the insertion of the catheter .

At the end of the surgical procedure, the animals received injections of analgesic (fentanyl, 0.032 mg/kg,
s.c.; Janssen Pharmaceutica, Beerse, Belgium) and prophylactic antibiotic (pentabiotic, 40,000 IU/kg, s.c.;
FortDodge, São Paulo, Brazil). The rats with difficulty in spontaneous urination had their bladders emptied
manually until they regained voiding function (generally in 12 h after surgery).

For this study, the animals were randomly divided into 4 groups: (1) Sham group, submitted to the mini-
laminectomy without the insertion of the catheter; (2) CTL-SCI group which was only submitted to the
spinal cord compression; (3) BV (NP)-SCI group received BV at non-acupuncture points at different time
points after the SCI; (4) BV (ST36 + GV3)-SCI group received BV injection at ST36 and GV3 acupoints at
[17]
different time points after the SCI .
BV solution preparation and treatments
BV (Apis mellifera, catalog #: V3375; Sigma, St. Louis, MO, USA) at a dose of 0.08 mg/kg was diluted in
saline solution and the application of BV solution was performed according to the group as previously
[17]
described . BV (ST36 + GV3)-SCI group received a subcutaneous injection of BV solution at acupoints
ST36 and GV3 (20 µL at each point). The acupoint ST36 is located approximately 5 mm below and lateral
to the anterior tubercle of the tibia, and GV3 is located on the dorsal midline at the depression between the
[30]
spinal processes of the last lumbar and the first sacral vertebrae . The BV (NP)-SCI group was injected
with the same dose and volume of BV at non-acupoints located in the same dermatome as the acupoints.