Purpose. To investigate whether acupuncture induced the proliferation and differentiation of endogenous neural stem cells (NSCs) in a rat model of traumatic brain injury (TBI). Methods. 104 Sprague-Dawley rats were randomly divided into normal, model, and acupuncture groups. Each group was subdivided into three-day (3 d), seven-day (7 d), and fourteen-day (14 d) groups. The rat TBI model was established using Feeney's freefall epidural impact method. The rats in the acupuncture group were treated at acupoints (Baihui, Shuigou, Fengfu, Yamen, and bilateral Hegu). The normal and model groups did not receive acupuncture. The establishment of the rat TBI model and the therapeutic effect of acupuncture were assessed using neurobehavioral scoring and hematoxylin-eosin staining. The proliferation and differentiation of NSCs in TBI rats were analyzed using immunofluorescence microscopy. Results. The levels of nestin-expressing cells and bromodeoxyuridine/glial fibrillary acidic protein- (BrdU/GFAP-) and BrdU/S100 calcium-binding protein B-positive and BrdU/microtubule-associated protein 2- and BrdU/galactocerebrosidase-positive cells were more significantly increased at various time points in the acupuncture group than in the model group (P < 0.01), except for a decreased level of BrdU/GFAP-positive cells at 7 d and 14 d. Conclusion. Acupuncture induced the proliferation and differentiation of NSCs, thereby promoting neural repair in the TBI rats.

ObjectiveTo study the effect of acupuncture on the TLR2/4-NF-κB signalling pathway in the cortex of Sprague-Dawley rats following traumatic brain injury (TBI), and investigate the possible mechanism underlying the effects of acupuncture on scar repair.MethodsTBI was established using Feeney’s free-falling epidural percussion model. In total, 108 rats were randomly divided into a normal group (n=18), untreated TBI model group (TBI group, n=36) and manual acupuncture-treated TBI group (TBI+MA, n=36). Each group of rats was subdivided into three time groups: 3-day (3d), 7-day (7d) and 14-day (14d). No treatment was given to rats in the normal and TBI groups. The TBI+MA group received manual acupuncture at GV20, GV26, GV16 through GV15, and bilateral LI4. mRNA expression of TLR2, TLR4, NF-κB and protein in the rat cortices was quantified using real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blot analyses.ResultsThe modified neurological severity score (mNSS) scores of the TBI+MA group were improved compared with baseline scores 12 hours after modelling, and improved at 7d and 14d compared with the TBI group (P<0.05), while the score of the TBI group did not improve until 14d compared to baseline. mRNA and protein expression of TLR2, TLR4 and NF-κB in the TBI group were higher than the normal group at 3d (P<0.05), reached a peak at 7d, then began to decrease at 14d. mRNA and protein expression of TLR2, TLR4 and NF-κB were higher in the TBI+MA group compared with the TBI group at 3d (P<0.05), were significantly down-regulated at 7d (P<0.01), and decreased to normal levels at 14d.ConclusionsAcupuncture has a bidirectional regulatory effect on the TLR2/4-NF-κB signalling pathway-related genes TLR2, TLR4 and NF-κB in the TBI rat cortex, promoting their expression in the early stage and inhibiting it in the later stage.

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.

Aims Recent investigations have already proved the neuroprotective efficacy of acupuncture in clinical practice in the treatment of neurological diseases, such as traumatic brain injury (TBI). Since growing evidence has suggested that neuronal autophagy was involved in multiple stages of TBI, this study aims to clarify the autophagy mediating mechanism underlying the neuroprotective effect of acupuncture in TBI rats. Methods Three experiments were carried out to detect changes in neuronal autophagy and identify the potential molecular mechanism underlying the neuroprotective effect of acupuncture for TBI treatment. Feeney's free‐falling epidural impingement method was used to establish the moderate TBI rat model; modified neurological severity scoring (mNSS) was used for neurological recovery evaluation. Nissl and HE staining were used to examine the histopathological changes. Immunofluorescence was used to detect the LC3‐positive cell rate. The transmission electron microscope (TEM) was used to investigate the morphology and quantity of autophagosomes. Western blotting was used to determine the protein expressions of LC3, p62, beclin1, mTOR, ULK1, p‐mTOR, and p‐ULK1. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used for gene expressions analysis of LC3 mRNA and p62 mRNA. Co‐immunoprecipitation (CO‐IP) method was used to identify the protein interaction of mTOR and ULK1. Results On Day 3 after TBI, acupuncture accelerated the removal of damaged cellular structures by promoting neuronal autophagy; on Day 7 and Day 14 after TBI, acupuncture inhibited neuronal autophagy, preventing excessive autophagy and thus alleviated nerve damage. In addition, the simultaneous treatment with 3‐MA or rapamycin at different stages after TBI attenuated the effect of acupuncture. Conclusion Acupuncture has a benign regulatory effect on neuronal autophagy in different stages of TBI, possibly through the mTOR/ULK1 pathway.

Objective: Neuroinflammation caused by traumatic brain injury (TBI) can lead to neurological deficits. Acupuncture can inhibit neuroinflammation and promote nerve repair; however, the specific mechanism is still unclear. The purpose of this study was to explore whether acupuncture could modulate the M1 and M2 phenotypic polarization of microglia in a rat model of TBI via the toll-like receptor 4 (TLR4)/intracellular toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)/myeloid differentiation factor 88 (MyD88) pathway. Methods: A total of 90 adult male Sprague-Dawley (SD) rats, SPF grade, were randomly divided into a normal group, model group and acupuncture group. Each group was further divided into three subgroups (first, third, and fifth day groups) according to the treatment time ( n = 10 rats/subgroup). We used the modified neurological severity score (mNSS) method to quantify neurological deficits before and after modeling. We used Nissl staining to observe the pathological changes in brain tissue, flow cytometry to detect the proportion of M1 and M2 polarized microglia in the injured area on the first, third and fifth day, and co-immunoprecipitation (Co-IP) to examine TLR4/TRIF/MyD88 expression in microglia on the first, third and fifth day, as well as expression of the amount of binding of TLR4 with TRIF and MyD88. Results: Compared to the model group, mNSS in the acupuncture group gradually decreased and pathological morphology improved. The proportion of CD11b/CD86 positive cells was decreased, while that of CD11b/CD206 was increased in the acupuncture group. Expression of IP TLR4, IP TRIF and IP MyD88 also decreased in the acupuncture group. Conclusion: The results of this study demonstrate that one of the mechanisms through which acupuncture mitigates neuroinflammation and promotes nerve repair in TBI rats may be inhibition of M1 phenotypic polarization and promotion of M2 phenotypic polarization through inhibition of the TLR4/TRIF/MyD88 signaling pathway.