Manual acupuncture relieves microglia-mediated neuroinflammation in a rat model of traumatic brain injury by inhibiting the RhoA/ROCK2 pathway

Abstract

Objective:

To investigate the regulatory mechanism of manual acupuncture (MA) on microglial polarization–mediated neuroinflammation after traumatic brain injury (TBI), focusing on the RhoA/Rho-associated coiled coil-forming protein kinase (ROCK2) pathway.

Methods:

Sprague Dawley (SD) rats were used to generate a TBI model using Feeney’s freefall epidural impact method. MA was performed on half of the TBI model rats, while the others remained untreated. Acupuncture was administered at GV15, GV16, GV20, GV26, and LI4. At the end of the intervention, rat brain tissue samples were collected, and the microglial M1 polarization status was observed by immunofluorescence labeling of CD86, an M1 microglia-specific protein. RhoA/ROCK2 signaling components were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors.

Results:

Compared with normal rats, the CD86 expression density in the untreated TBI model rats was high and showed an aggregated expression pattern. The genes and proteins of the RhoA/ROCK2 signaling pathway were highly expressed, and inflammatory factors were significantly increased. The CD86 expression density in TBI rats after MA was reduced compared to that in untreated TBI rats and showed a scattered distribution. The expression of RhoA/ROCK2 signaling pathway genes and proteins was also significantly reduced, and inflammatory factors were decreased.

Conclusion:

These results show that MA may inhibit M1 polarization of microglia by regulating the RhoA/ROCK2 signaling pathway, thereby reducing neuroinflammation in TBI.

Keywords

manual acupuncture microglia neuroinflammation RhoA/ROCK2 signaling pathway traumatic brain injury

Introduction

Neuroinflammation is the most obvious pathology following traumatic brain injury (TBI) and is the leading cause of secondary injury. Neuroinflammation is closely related to microglia and astrocytes. Microglia are one type of primary innate immune response cell in the brain¹ and can stabilize the internal environment.² Two types of microglial polarization have been observed to date: M1 and M2.³ In the early stages of injury, microglia typically undergo M1 polarization and secrete high levels of inflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α to initiate immune defense.⁴ However, if inflammatory stimulation persists, an excessive M1 polarization response instead leads to excessive release of pro-inflammatory factors and neurotoxic mediators; this results in neurotoxicity and causes a vicious cycle of microglia-mediated neurodegenerative damage.⁵

Microglial M1 polarization leads to the secretion of IL-1β, IL-6, TNF-α, and other inflammatory factors.⁶ It may be directly related to the RhoA/Rho-associated coiled coil-forming protein kinase 2 (ROCK2) signaling pathway. One study showed that inhibition of the ROCK2 pathway significantly reduced the secretion of inflammatory factors from microglia,⁷ thereby inhibiting neuroinflammation caused by central nervous system injury. The RhoA/ROCK2 signaling pathway mediates inhibitory signals that block cellular regeneration in the central nervous system.⁸ In TBI, microglia express RhoA/ROCK2, causing neuroinflammation.⁹

Acupuncture effectively inhibits neuroinflammation after TBI, reduces symptoms of cerebral edema, improves patient awakening and survival rates, and improves symptoms of sequelae. These treatment effects have been verified clinically.^10,11 However, the mechanisms underlying acupuncture treatment have not yet been fully elucidated. To explore the effects and mechanisms of acupuncture treatment following TBI, we used Sprague Dawley (SD) rats to construct a TBI model, applied manual acupuncture (MA) as an intervention, and conducted a series of basic research experiments. In a previous study, we found that astrocytes were activated in the early post-TBI period and aggregated to form glial scars. Excessive formation of glial scars affects nerve repair and aggravates neuroinflammation. MA inhibits the excessive formation of glial scars after TBI in rats, thus inhibiting neuroinflammation.¹² Microglia are a component of glial scars. Moreover, neuroinflammation after TBI is accompanied by the activation and polarization of microglia. Excessive M1 polarization of microglia causes intense neuroinflammation. In addition, we also previously found that MA regulates the expression of Nogo-A in the serum of TBI rats, thereby slowing nerve damage. Nogo-A activates RhoA guanosine triphosphate (GTP)ase through the Nogo receptor, which then activates the effector ROCK2 to mediate the inhibition of neurite outgrowth.¹³ In this study, we aimed to address whether MA regulates the M1 polarization of microglia to alleviate neuroinflammation through regulation of the RhoA/ROCK2 signaling pathway.

Methods

Generation of a TBI model

A total of 30 adult male SD rats weighing 250 ± 20 g were purchased from Guangdong Medical Laboratory Animal Center (license no. SCXK (Guangdong) 2013-0002; experimental animal certificate no. 44007200034600). The animal experiments were conducted at the Experimental Animal Management Center of Jinan University (license no. SCXK (Guangdong) 2012-0117).

After 1 week of adaptive feeding, SD rats were divided into the following three groups using a random number table method: TBI + MA group; TBI group; and normal group. There were 8 animals in the normal group and 11 animals each in the TBI + MA and TBI groups. Because the study required construction of a model with moderate to severe craniocerebral injury, some experimental rats were killed during the modeling process. Dead TBI rats were excluded, and the TBI + MA group and TBI group retained only eight rats each per group after generating the model.

For the TBI group and the TBI + MA group, Feeney’s freefall epidural impact method was used to construct a rat model of moderate craniocerebral injury.¹² The rats were weighed and anesthetized via an intraperitoneal injection of 30 mg/mL pentobarbital sodium (45 mg/kg). After anesthesia, the scalp was cut 2 mm to the right of the skull median line and separated from the skull. The skull was drilled with a flexible skull drill to open a round window with a diameter of 5 mm at a position 2 mm to the right of the sagittal suture and 1 mm posterior to the coronal suture; the dura was kept intact. A 20 g hammer was allowed to fall freely from a height of 30 cm and to hit the flat nail placed on the window of the skull, resulting in local cerebral contusion of the right parietal lobe. The scalp was sutured after full hemostasis and, finally, the sutured skin was wiped with 75% alcohol. The normal group did not receive any surgical treatment.

Acupuncture intervention

Twenty-four hours after surgery, the rats that died were removed, and eight surviving rats were retained in both the TBI group and the TBI + MA group. Rats in the TBI + MA group then immediately received the MA intervention. The rats were fixed on a fixation frame, then 0.18*13 mm filiform stainless-steel needles were inserted at GV15 (Yamen), GV16 (Fengfu), GV20 (Baihui), GV26 (Shuigou), and LI4 (Hegu) according to traditional acupuncture point localization of rats described in “Experimental Acupuncture.”¹⁴ During the operation, each needle was advanced to a depth of 2 mm and manipulated with a twisting method. The twisting range was 360°, and the frequency was 120–160 beats/min. The operation lasted for 1 min per needling site and the needle remained in place for 15 min, during which time it was twisted once every 5 min. The MA treatment was conducted once a day. The rats in the normal and TBI groups were not treated, but they were fixed for 15 min to ensure that they were exposed to the same handling and fixation conditions as the TBI + MA group. The MA treatment lasted for 3 days.

Tissue sampling

Sample collection from all rats in all of the groups was performed 1 h after the last acupuncture intervention. Rats were anesthetized with pentobarbital sodium (at the dose previously described) and decapitated, and cold physiological saline was used to infuse the brain. Once the intact brain was removed, the cortical tissue surrounding the lesion was collected (see Supplemental Material). Then, the cortical tissue samples were processed using the appropriate methods for each detection protocol.

Immunofluorescence labeling

The cortical tissue surrounding the lesion was fixed for 24 h and dehydrated with a gradient of sucrose solutions. The dehydrated tissue was placed in optimal cutting temperature (OCT) embedding agent, after which the tissue was transferred to a cryostat for quick freezing and embedding. Then, the tissue was sectioned at a thickness of 10 µm. The cut frozen tissue sections were fixed in cold acetone and rinsed with phosphate buffer saline (PBS). The sections were incubated in 0.3% Triton X-100 (diluted with bovine serum albumin (BSA)) to increase cell permeability. A working solution (3% BSA) was used for blocking. A diluted (1:200) CD86 (an M1 microglia-specific protein) antibody (aho1057317, Bioss, China) was added to the section and incubated at 4°C overnight. After rinsing, a suitable dilution (1:400) of fluorescent goat anti-mouse IgG secondary antibody (ab150115, Abcam, United Kingdom) was added to the section and incubated at room temperature for 1–2 h. The section was rinsed with PBS, and 4ʹ,6-diamidino-2-phenylindole (DAPI) staining solution (Ab104140, Abcam) was added dropwise to the section before an 8 min incubation in the dark for nuclear labeling. The section was washed with PBS, and the slide was sealed with antifade mounting medium. Sections were observed under a fluorescent microscope, and images were taken. The positive area rate was calculated using Image J.

Quantitative real-time polymerase chain reaction

Quantitative fluorescence polymerase chain reaction (PCR) (SYBR Green method) was used to detect the expression levels of RhoA and ROCK2 in the cortical tissue sample surrounding the lesion. An appropriate amount of cortical tissue was removed, and TRIzol (Invitrogen, Waltham, MA, United States) was used to extract the total RNA using a standard method. The RNA was purified with RNase-free DNase I (Promega, Madison, WI, United States). The optical density (OD) value was measured with an ultraviolet spectrophotometer (UV-1750, Shimadzu, Japan) to assess the RNA purity, and gel electrophoresis was performed to assess RNA integrity. The PrimeScript™ Reverse Transcription Kit (RR047A, TaKaRa, Japan) was used for reverse transcription. Primer synthesis was performed by Sangon, Shanghai, using an ABI 3900 high-throughput DNA synthesizer. The sequences of the synthesized primers are listed as follows: RhoA (101 bp) forward 5ʹ-ATTGGCGCTTTTGGGTACAT-3ʹ reverse 5ʹ-CCGCGTCTAGCTTGCAGAG-3ʹ; ROCK2 (98 bp) forward 5ʹ-TGGAGTACATGCCAGGTGGA-3ʹ reverse 5ʹ-TGCAAGCACGACTTCAGCA-3ʹ; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (94 bp) forward 5ʹ-ATTCCACCCACGGCAAGTT-3ʹ reverse 5ʹ-TCTCGCTCCTGGAAGATGGT-3ʹ. A SYBR Premix EX Taq II Kit (RR820A, TaKaRa) was used for the fluorescent PCR reactions, and the reaction protocol was as follows: 95°C for 3 min; 95°C for 5 s; 60°C for 30 s; 40 cycles of 95°C for 15 s and 60°C for 30 s; and 95°C for 15 s. After the reaction, the 2^−ΔΔCT relative quantitative method was used for the analysis.

Western blot analysis

Western blotting was used to detect expression of RhoA and ROCK2. Brain tissue from the cortex near the lesion was removed. After total protein was extracted, 80 µL of sample was mixed with 20 µL of 5× sample treatment solution. The tube containing the mixture was sealed and boiled in a 100°C water bath for 10 min. Then, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Proteins in the 10% separation gel were transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked for 30 min with 5% non-fat milk. Primary antibodies RhoA rabbit anti-rat (Ab86297, Abcam) or ROCK2 rabbit anti-rat (Ab71598, Abcam) were added at a dilution of 1:1000, and the membrane was incubated at 4°C overnight. GAPDH was used as an internal reference (1:1000, rabbit anti-rat; Ab9484, Abcam). After rewarming at 37°C for 30 min, the membrane was washed three times in Tris-buffered saline with Tween 20 (TBST) and then incubated with secondary antibody (1:4000, goat anti-rabbit IgG-HRP; sc-2004, Santa Cruz Biotechnology, United States). After washing with TBST, an enhanced chemiluminescence (ECL) agent was added. The sample was developed and exposed in a Sage Creation gel imaging system.

Enzyme-linked immunosorbent assay

Cortical samples near the lesion were prepared as 10% tissue homogenates. Rat brain tissue was rinsed in 4°C pre-chilled PBS to remove blood, and the residual PBS was absorbed by filter paper. The brain was weighed, and lysis homogenization buffer was added (on ice). The resultant homogenate was repeatedly frozen and thawed using liquid nitrogen three times and was allowed to stand at room temperature for 30 min. The homogenate was centrifuged at 250 × g for 1 h at 4°C, and the supernatant was saved. Enzyme-linked immunosorbent assay (ELISA) for IL-1β, IL-6, and TNF-α was then performed in strict accordance with the manufacturer’s instructions (JYM0419Ra, JYM0646Ra, and JYM0635Ra, respectively, ColorfulGene, China).

Data analysis

Results are expressed as mean ± SD. Data were analyzed using STATA 11.0 (StataCorp, United States). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Scheffe test for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

Results

Effect of acupuncture on CD86 expression

CD86 is a specific marker protein of microglial M1 polarization. Using immunofluorescent labeling of CD86, we evaluated the M1 polarization status and inflammatory state of microglia in each group of specimens. We used red fluorescence to label CD86 and blue fluorescence to label the nuclei. As shown in Figure 1(a), only a small amount of red fluorescence was detected in the normal group, indicating that the microglia had only a small amount of M1 polarization in normal brain tissue. The density of red fluorescence in the TBI group was high and showed a clustered distribution; moreover, the positive areas in individual cells were also large, indicating that CD86 was highly expressed. This suggests a high degree of M1 polarization and a robust inflammatory response. The red fluorescence density of the TBI + MA group was reduced compared to the TBI group, and the distribution was mostly scattered and not clustered like in the TBI group. The positive fluorescent area for the TBI group was significantly higher than that for the normal group (P < 0.001; Figure 1(b)). For the TBI + MA group, which received acupuncture intervention, the positive fluorescent area was significantly smaller than that for the TBI group (P < 0.001).

Effect of acupuncture on RhoA/ROCK2 gene expression

The results in Figure 2 show that the expression levels of RhoA and ROCK2 followed similar trends. The TBI group had significantly higher expression levels of the RhoA gene than did the normal group (P < 0.001; Figure 2(a)), and the TBI + MA group had significantly lower RhoA expression than did the TBI group (P < 0.01). The TBI group had significantly higher expression levels of ROCK2 than did the normal group (P < 0.01; Figure 2(b)), and the TBI + MA group had significantly lower expression levels of ROCK2 than did the TBI group (P < 0.05).

Effect of acupuncture on RhoA/ROCK2 protein expression

Western blot analysis (Figures 3(a) and 4(a)) showed that the positions of the bands for RhoA, ROCK2, and the internal GAPDH control were roughly the same for each sample, the bands were clear, and the internal control expression levels were consistent. The relative gray value of the RhoA protein in the TBI group was significantly higher than that in the normal group (P = 0.003 < 0.01; Figure 3(b)). Compared with the relative gray value of RhoA in the TBI group, that in the TBI + MA group was significantly lower (P < 0.05). The comparisons of relative gray values of ROCK2 were roughly the same as those for RhoA (Figure 4(b)). Compared with the relative gray value of ROCK2 in the normal group, that in the TBI group was significantly higher (P < 0.01). Compared with the relative gray value of ROCK2 in the TBI group, that in the TBI + MA group was significantly lower (P < 0.01).

Effects of acupuncture on the expression of IL-1β, IL-6 and TNF-α

The expression levels of the inflammatory factors IL-1β, IL-6, and TNF-α (Figure 5) followed similar trends. Compared with the expression of inflammatory factors in the normal group, that in the TBI group was higher (P < 0.001). Compared with the expression of inflammatory factors in the TBI group, that in the TBI + MA group was significantly lower (P < 0.001).

Discussion

M1-type microglia have many surface antigens, such as CD86, MHC-II, CD16, and CD32.¹⁵ However, CD86 is the most used and easily labeled marker for M1-type microglial detection.¹⁶

From the CD86 immunofluorescence labeling, we observed that rats in the TBI group had an increased density of positive red fluorescence in the cortex surrounding the brain tissue lesions on the third day after brain trauma (compared with the normal group) and the fluorescence showed a clustered distribution. This suggests that microglia surrounding the lesion had increased M1-type polarization after the brain injury. In addition, the expression analysis showed that the TBI group had significantly elevated gene and protein levels of RhoA and ROCK2 (compared with the normal group) and that levels of the inflammatory factors IL-1β, IL-6, and TNF-α were also significantly higher than in the normal group. This indicated that the RhoA/ROCK2 signaling pathway was activated in rats after brain injury and M1 polarization occurred in microglia. This polarization increased the expression of downstream inflammatory factors, including IL-1β, IL-6, and TNF-α, which reflects severe neuroinflammation in the brain. The rats in the TBI + MA group started receiving MA intervention on the first day after brain injury and various indicators were different from those in the TBI group. In the TBI + MA group, CD86 immunofluorescence density decreased (compared with the TBI group) and it was relatively scattered. This suggests that M1 activation in microglia surrounding the brain lesions in TBI rats was reduced after MA intervention. The gene and protein levels of RhoA and ROCK2 in the TBI + MA group also decreased (compared with the TBI group) and the levels of the inflammatory factors IL-1β, IL-6, and TNF-α were significantly lower than those in the TBI group. This result indicates that MA intervention may inhibit the expression of the RhoA/ROCK2 signaling pathway in the cortex surrounding the lesions in TBI rats, causing reduced M1 polarization of microglia and dampened levels of the downstream inflammatory factors IL-1β, IL-6, and TNF-α; these effects could ultimately improve neuroinflammation status.

Acupuncture therapy, which has features of traditional Chinese medicine, is considered to be an effective treatment for TBI. It has been proposed that its efficacy may be closely associated with needling location, that is, the selection of traditional acupuncture points. In this study, the traditional acupuncture point group prescription was primarily composed of GV points, including GV15, GV16, GV20, and GV26. Our previous clinical trials showed that a similar traditional acupuncture point prescription can effectively promote recovery in TBI patients.¹⁷ A literature search also suggested that GV points may have unique efficacy in the treatment of brain diseases, although the neurophysio-logical basis of this observation has not been fully elaborated.^18–20

In our previous study, we used neurobehavioral observation and hematoxylin–eosin (HE) staining to determine whether MA improved brain injury in TBI rats.²¹ Neurobehavioral observation revealed that the TBI + MA group scores on the 7th and 14th day began to improve, and they differed significantly from the untreated TBI group scores (P < 0.05). The HE staining also showed that TBI rats without treatment had more significant pathological changes and slower recovery than TBI rats receiving acupuncture treatment on the 7th and 14th day. Neither the neurobehavioral scores nor the HE staining were significantly different on the 3rd day in the TBI + MA group compared to TBI group (P > 0.05).

Although the neurobehavior and brain tissue changes in the later stages of recovery from TBI, neuroinflammation is usually severe during the early stages, responding violently 1–3 days after the onset of TBI and gradually stabilizing during later stages.²² Therefore, in this study, we designed a 3-day experimental cycle to observe the effects of MA on the polarization of microglia, the primary carrier cells of neuroinflammation, after early intervention. Neuroinflammation is the most obvious pathology following TBI, and the polarization status and levels of microglia near the damaged brain area affect its progression. Excessive M1-type polarization of microglia causes severe local neuroinflammation. This mechanism is generally believed to be associated with the RhoA/ROCK2 signaling pathway. RhoA belongs to the Rho family and is a GTPase. When microglia receive stimulation from damaged neurons and pro-inflammatory factors, they activate RhoA and its downstream effector kinase ROCK, which exists in two subtypes (ROCK1 and ROCK2). ROCK2 is primarily present in the nerve center and is highly expressed.²³ ROCK2 plays a key role in inhibiting central nervous system growth and is activated by multiple inhibitory receptors; therefore, the inhibition of ROCK2-related signaling pathways is important for blocking nerve growth inhibition following TBI and improving neuroinflammation.

Our previous study found that acupuncture effectively improves the modified neurological severity score (mNSS) of TBI rats; this score is an evaluation index of neurological functional recovery.¹² In addition, acupuncture reduces the formation of glial scars by reducing astrocyte aggregation. Microglia are also involved in glial scar formation. This study shows that MA effectively reduces M1 polarization of microglia, which is consistent with previous conclusions. However, we also previously found that MA promotes the recovery of nerves by promoting growth and differentiation of neural stem cells.^21,24 This study confirms that MA has a probable inhibitory effect on the RhoA/ROCK2 signaling pathway. Because ROCK2 inhibits nerve growth, it is reasonable that nerve recovery improvements following acupuncture intervention for TBI are also related to ROCK2-related signaling pathway inhibition. Whether it acts by reducing neuroinflammation and blocking secondary injury of the central nervous system or by protecting the internal environment of the central nervous system and promoting recovery of damaged nerves, the inhibition of the ROCK2-related signaling pathway has potential therapeutic significance. Without doubt, we need to conduct further experiments inhibiting the RhoA/ROCK2 pathway to further illustrate its role in MA. Thus, we will conduct a deeper investigation of the interactions between MA and ROCK2-related upstream and downstream signals.

Conclusion

Taken together, these results suggest that MA effectively inhibits M1 activation of microglia in the cortex surrounding the lesions in TBI rats and reduces the expression of inflammatory factors, thereby inhibiting neuroinflammation and promoting nerve repair. This effect is closely associated with inhibition of the RhoA/ROCK2 signaling pathway.

Supplemental Material

AIM912248_Supplemental_material – Supplemental material for Manual acupuncture relieves microglia-mediated neuroinflammation in a rat model of traumatic brain injury by inhibiting the RhoA/ROCK2 pathway

Supplemental material, AIM912248_Supplemental_material for Manual acupuncture relieves microglia-mediated neuroinflammation in a rat model of traumatic brain injury by inhibiting the RhoA/ROCK2 pathway by Ming-min Zhu, Ji-huan Lin, Peng Qing, Liu Pu, Shu-lian Chen, Shu-jun Lin, Cheng-lu Li, Lu-xi Cao and Yi-min Zhang in Acupuncture in Medicine

Footnotes

Contributors

Experiments were designed by M-Z and P-Q and performed by M-Z and J-L Y-Z and P-Q provided guidance. L-P, S-C, and S-L assisted with the experiments. C-L and L-C analyzed the data. Y-Z wrote the manuscript, and all authors approved the final version accepted for publication. M-Z, J-L and contributed equally in this study. M-Z, J-L and P-Q contributed equally in this study.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by National Natural Science Foundation of China (grant no. 81704156), Natural Science Foundation of Guang-dong Province (grant no. 2016A030310093 and 2017A030310024), Administration of Traditional Chinese Medicine of Guangdong Province, China (grant no. 20161067 and 20181070), and the Fundamental Research Funds for the Central Universities, China (grant no. 21616318).

Ethical approval

This experiment was performed in strict accordance with the Regulations on the Administration of Experimental Animals issued by the State Council of China in 1988 and “3R” principles of Replacement, Reduction, and Refinement. In addition, the experiments were approved by the Laboratory Animal Ethics Committee of Jinan University (batch no. 20160922105817). All rats were subjected to general anesthesia during TBI model generation via intraperitoneal injection of 30 mg/mL pentobarbital sodium (45 mg/kg). All measures were taken to minimize the pain of the experimental animals.

Provenance and peer review

This research was not commissioned; externally peer reviewed.

ORCID iD

Yi-min Zhang

Supplemental material

Supplemental material for this article is available online.

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