Acupuncture, one of the therapies of Traditional Chinese Medicine based on the stimulation of specific body points[
BV is a complex substance with approximately 18 bioactive components, including phospholipase A2, histamine, norepinephrine, apamin and melittin (its principal component, representing 40%-60% of the BV total dry extract)[
When BV is applied in acupoints, it promotes intensification of its effects and more intense and lasting acupoint stimulation effects[
Previous results from our group, using a compression SCI model, showed that apipuncture in ST36 and GV3 acupoints was able to modulate the balance between pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines, promoting a reduction of spinal cord tissue loss and improvement of locomotor performance[
The profile of microglia/macrophage polarization phenotypes in M1/M2 are initially simplified paradigms in an attempt to understand the complexity of the inflammatory response[
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.
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 previously described by Vanický
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 different time points after the SCI[
BV (
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.
For the evaluation of the locomotor capacity, the animals were submitted to the Basso, Beattie, and Bresnahan (BBB) test as described previously[
Based on prior publications[
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
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, 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).
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
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
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,
Apipuncture improves locomotor performance in SCI rats. The results of apipuncture at GV3 and ST36 acupoints [BV (ST36 + GV3)-SCI,
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 test;
Influence of apipuncture in the mRNA expression of M1 (iNOS) and M2 (TGF-β and Arg-1) phenotype markers and COX-2 in the spinal cord 1, 3 and 5 days after SCI. The graph represents the iNOS (A), TGF-β (B), Arg-1 (C) and COX-2 (D) mRNA expression at the site of spinal cord injury in rats submitted to SCI and apipuncture at ST36 and GV3 points [BV (ST36)-SCI;
The group BV (ST36 + GV3)-SCI showed significant lower levels of IBA-1 (microglia/macrophage active marker) protein compared to CTL-SCI and BV (NP)-SCI groups at all times evaluated (1st, 3rd and 5th days) after SCI (One way ANOVA followed by Bonferroni test;
Influence of apipuncture on IBA-1 levels in the spinal cord 1, 3 and 5 days after SCI. In A: representative of the bands of proteins of IBA-1 by the groups Sham, CTL-SCI, BV (NP)-SCI and BV (ST36 + GV3)-SCI measured by densitometry, normalized by β-actin; In B: the comparison of the IBA-1 protein content at the spinal cord lesion site of rats submitted to SCI and apipuncture at ST36 and GV3 points [BV (ST36)-SCI;
The group BV (ST36 + GV3)-SCI showed significant higher levels of BCL-2 (anti-apoptotic factor marker), compared to CTL-SCI and BV (NP)-SCI groups on the 5th day after SCI (One way ANOVA followed by Bonferroni test;
Influence of apipuncture on BCL levels in the spinal cord 1, 3 and 5 days after SCI. In A: representative of the bands of proteins of BCL-2 by the groups Sham, CTL-SCI, BV (NP)-SCI and BV (ST36 + GV3)-SCI measured by densitometry, normalized by β-actin; In B: the comparison of the BCL-2 protein content at the spinal cord lesion site of rats submitted to SCI and apipuncture at ST36 and GV3 points [BV (ST36)-SCI;
Influence of apipuncture on NeuN and CNPase levels in the spinal cord 7 days after SCI. In A: representative of the bands of proteins of NeuN and CNPase by the groups Sham, CTL-SCI, BV (NP)-SCI and BV (ST36 + GV3)-SCI measured by densitometry, normalized by β-actin; In B and C: the comparison of the NeuN and anti-CNPase protein content at the spinal cord lesion site of rats submitted to to SCI and apipuncture at ST36 and GV3 points [BV (ST36)-SCI;
Our results demonstrated that apipuncture treatment promoted improvement in locomotor function in the SCI compression model in rats. This improvement seems to be associated with neuroinflammation modulation through the reduction of the microglia/macrophage protein marker IBA-1, changes in the expression of M1 (iNOS) and M2 gene markers (TGF-β and ARG-1), and decrease of COX-2 expression. Furthermore, apipuncture increased protein levels of the anti-apoptotic marker BCL-2, as well as NeuN and CNPase markers indicating the reduction of the death of neurons and oligodendrocytes at the site of SCI.
The previous study conducted by our group, using the same experimental protocol showed that BV acupuncture at ST36 and GV3 acupoints was able to improve locomotor performance, reduced the area of tissue damage in the spinal cord and modulate the balance between cytokines by increasing the anti-inflammatory cytokine IL-10 and reducing the proinflammatory cytokine IL-6[
In the acute phase of the inflammatory response following SCI, M1 polarization phenotype markers are prevalent and pro-inflammatory factors such as IL1β, IL-6, TNF-α and iNOS contributing to positive feedback that triggers an exaggerated inflammatory response causing greater tissue damage around the SCI area[
Previous studies have reported that the use of drugs, such as steroidal anti-inflammatory, that block the acute inflammatory response leads to more neurological damage in the SCI model than when this blockade does not occur[
After the injury, an inflammatory response is initiated, in which microglia/macrophages quickly become active by chemical signals released by neural cell death[
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[
In the present study, the apipuncture treatment was able to reduce COX-2 mRNA on the 3rd day after SCI. Although, in some tissues like the brain and the spinal cord COX-2 is constitutively expressed[
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[
Despite the limitation of study in not having immunohistochemical analyses, the increase of anti-apoptotic factor BCL-2 caused by apipuncture can be related to neuroprotection and survival of neurons and oligodendrocytes resulting in better sensory and locomotor performance. Choi
The clinical practice of different acupuncture modalities, such as BV acupuncture and EA, has become increasingly used as a complementary therapy for symptom relief in patients with PD, ALS, and SCI[
The exact mechanism involved in the anti-inflammatory effects of acupoint stimulation has not yet been completely elucidated. Currently, it has been postulated the stimulation of acupuncture points acts through the autonomic nervous system, transmitting signals via the vagus nerve and promoting anti-inflammatory responses[
In conclusion, despite some limitations, our results indicate that apipuncture may modulate the neuroinflammatory response via alteration of M1/M2 polarization status, in addition to increasing the apoptotic factor and promoting neuroprotection, which may in part contribute to a reduction in locomotor sequelae in the compression SCI model. Thus, we believe that apipuncture may be a potential therapeutic target as a complementary therapy for the treatment of spinal cord injury.
We are indebted to Mr. Ipojucan Pereira de Souza for technical assistance.
Made substantial contributions to conception and design of the study: Souza RN, Medeiros MA
Performed data analysis and interpretation: Souza RN, Monteiro LRN, Medeiros MA.
Performed data acquisition, as well as provided administrative, technical, and material support: Souza RN, Lopes JMA, Monteiro LRN, Barbosa RAQ, Hollmann G, Allodi S, Reis LC, Medeiros MA
Not applicable.
This work was supported by FAPERJ (Research support foundation in the state of Rio de Janeiro) (grand number: 111.616/2010).
All authors declared that there are no conflicts of interest.
All procedures were approved by the Ethics Committee on Research of the Federal Rural University of Rio de Janeiro (23083.005880/2013).
Not applicable.
© The Author(s) 2019.