Page 130 - Read Online

Souza et al. Neuroimmunol Neuroinflammation 2019;6:12 I http://dx.doi.org/10.20517/2347-8659.2019.04 Page 9 of 13

as well as in other neuroinflammatory diseases, there is deregulation between these states of polarization,
causing greater death of neurons and oligodendrocytes due to greater initial activation of more cytotoxic
status (M1) followed by a later activation of neuroprotective states (M2) [18,21,25,26] . Thus, it is believed that
therapies that promote total blockade of the inflammatory response could inhibit both M1 and M2 status
thus hampering tissue repair and aggravating damage after SCI [20,25] . Our data have shown that apipuncture
was able to influence the reduction of iNOS, linked to M1 status and the increase of TGF-b and Arg-1,
linked to M2 status, thus suggesting that just as EA, apipuncture treatment might modulate inflammation
via M1/M2 status.

After the injury, an inflammatory response is initiated, in which microglia/macrophages quickly become
active by chemical signals released by neural cell death [36,37] , they are considered as one of the main
initiators of the chronic response, triggering greater secondary damage [18,25,34] . When active, they express
the ionized calcium-binding adapter molecule-1 (IBA-1) which has been widely used as a polarized
[38]
microglia/macrophage marker .

Our data revealed significantly increased IBA-1 levels in the groups submitted to the surgical procedure
compared to the sham group in the first 24 h, which remained increased until the 5th day. However,
treatment with apipuncture at acupoints ST36 and GV3 significantly reduced the IBA-1 marker compared
to control groups. This modulation in the IBA-1 levels is important in the reduction of neuroinflammation
since after SCI there is an exacerbated neuroinflammatory response with microglia/macrophages
activation [20,36,39,40] , causing greater tissue damage, with the death of neurons and oligodendrocytes in the
[8]
first hours after the trauma [18,25,41] . Similar results were shown by Kang et al. using the SCI model followed
by apipuncture treatment at ST36 which was able to reduce the IBA-1 marker detected by Western blotting.
Moreover, the Kang and colleagues’ study, the expression of the IBA-1 marker was very low in the sham
[8]
group . Manual acupuncture at ST36 acupoints can also contribute to the modulation of the inflammatory
response due to a reduction of the IBA-1 marker in the ALS model using hSOD1 G93A animals . Our results
[3]
suggest that apipuncture reduces microglia/macrophage polarization, but it is noteworthy that it is likely
to be more involved with M1 status as it additionally reduced iNOS mRNA expression. More studies are
needed to enhance these results.

In the present study, the apipuncture treatment was able to reduce COX-2 mRNA on the 3rd day after SCI.
[42]
Although, in some tissues like the brain and the spinal cord COX-2 is constitutively expressed after SCI
[4]
occurs induction of COX-2 , which increases mainly prostaglandin PGE2 that binds to prostaglandin
E receptor subtypes in endothelial cells. It also stimulates the activation of NF-κB and consequently the
nuclear transcription of chemokines such as MIP-1, attracting hematogenous macrophages to the lesion
site. It is believed that this mechanism may contribute to a chronic inflammatory response in some
situations [42,43] . Corroborating with our results, it has been reported that manual acupuncture reduces COX-
2 expression 24 h after SCI and that EA also reduces COX-2 expression in the neuropathic pain model [4,44] .

In the present study, apipuncture at ST36 + GV3 also increased BCL-2 levels on the 5th after SCI.
Additionally, on the 7th day after SCI, apipuncture significantly minimized the reduction of NeuN protein
content (a neuron marker) and of CNPase, an enzyme expressed by viable oligodendrocytes, indicating a
lower death of these cell types. Previous studies also indicated that acupuncture and electroacupuncture
were able to increase BCL-2 protein levels and reduce BAX and caspase-3 levels, maintaining a higher
number of viable neurons after SCI [4,45] . After SCI, there is also an imbalance between pro and anti-
apoptotic proteins. BAX is a pro-apoptotic protein that stimulates mitochondrial damage through the
formation of pores in the mitochondrial membrane, releasing cytochrome C (Cyt-C) which stimulates the
cleavage of caspase-3, an enzyme that leads to cell death [46,47] . BCL-2 is an important anti-apoptotic protein
that acts by modulating BAX and reducing the stimulation of intrinsic apoptotic factors [48,49] . Corroborating