Electroacupuncture alleviates neuropathic pain caused by SNL by promoting M2 microglia polarization through PD-L1.

As a common clinical disease, neuropathic pain is difficult to be cured with drugs. The occurrence and progression of pain is closely related to the response of spinal microglia. Aspartof the regulation of microglialactivity,PD-L1 playsacriticalrole. Loss of PD-L1 promoted the polarization of M1-like microglia. Increased expression of PD-L1 promoted M2-like polarization. Electroacupuncture has a significant analgesic effect in clinical practice, but its specific mechanism remains to be further explored. In this study, we verified the role of PD-L1 in EA analgesia and the underlying molecular mechanism through spinal nerve ligation (SNL) in rats and lipopolysaccharide (LPS)-treated BV2 microglial cells. Forbehavioralstudiesofrats,mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured, and spinal cord neuros were examined under transmission electron microscopyto determine changes to their myelin structure. The expression levels of PD-L1 and M1/M2-specific markers in rat spinal cord and BV2 microglial cells were measured by enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence staining and Western blot analysis. Our study showed that EA increased the pain threshold, reduced the destruction of myelin structure, promoted the expression of PD-L1 and PD-1, inhibited the MAPK signaling pathway, and promoted the conversion of microglial polarization from the M1 phenotype to the M2 phenotype in SNL rats. PD-L1 knockdown reversed these effects of EA. In addition, PD-L1 knockdown activated the MAPK signaling pathway, promoted microglial polarization to the M1 phenotype, decreased the expression of anti-inflammatory mediators and increased the expression of proinflammatory factors in LPS-stimulated BV2 microglial cells. Our results showed that EA may regulate the excitability of primary afferent neurons through PD-L1 and then inhibit the MAPK signaling pathway to promote the transformation of activated M1 microglia into M2 microglia, reduce inflammatory reactions, and finally achieve analgesic effects. A therapy targeting PD-L1 may be an effective strategy for treating neuropathic pain.

Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL.

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Electroacupuncture Alleviates Neuropathic Pain through Regulating miR-206-3p Targeting BDNF after CCI.

Background: Electroacupuncture (EA) has benefits for neuropathic pain. However, the underlying mechanisms are still unknown. The current study explores the underlying mechanisms of EA in neuropathic pain of chronic constriction injury (CCI) rats. Material/Methods. Overall, 126 Sprague-Dawley (200-250 g) rats were divided into nine groups randomly: the sham-operated, CCI, CCI+EA, CCI+sham EA, CCI+NS, CCI+AAV-NC, CCI+AAV-miR-206-3p, CCI+EA+NS, and CCI+EA+AAV-miR-206-3p groups. The animals were sacrificed 14 days postsurgery. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests were used to determine differences in neurobehavioral manifestations. qPCR, western blotting, and immunofluorescence (IF) were carried out to detect the expression levels of miR-206-3p, BDNF, BAX/Bcl-2, TNF-α, and IL-6. Nissl staining was measured to observe morphological changes in neurons. Transmission electron microscopy (TEM) was employed to evaluate microscopic changes in dorsal horn synapses. Results: Hyperalgesia was reduced markedly by EA in the CCI model. The expression level of miR-206-3p was elevated, whereas the expression levels of BDNF, BAX/Bcl-2, TNF-α, and IL-6 were decreased in EA-treated CCI rats. However, a miR-206-3p inhibitor partially abrogated the analgesic effect of EA and resulted in poor behavioral performance and the BDNF, BAX/Bcl-2, TNF-α, and IL-6 expression was elevated as well. Conclusions: EA can relieve neuropathic pain by regulating the miR-206-3p/BDNF pathway, thus exerting anti-inflammatory and antiapoptotic effect.

Electroacupuncture suppresses spinal nerve ligation-induced neuropathic pain via regulation of synaptic plasticity through upregulation of basic fibroblast growth factor expression.

BACKGROUND: Improving synaptic plasticity is a good way to alleviate neuropathic pain. Electroacupuncture (EA) is currently used worldwide to treat this disease, but its specific mechanisms of action need further investigation. Evidence has suggested that basic fibroblast growth factor (bFGF) plays an important role in promoting nerve regeneration and can promote the expression of vascular endothelial growth factor (VEGF). OBJECTIVE: In this study, we examined the effects of EA on synaptic plasticity and its underlying mechanism. METHODS: A spinal nerve ligation (SNL) rat model was established. NSC37204 (a specific inhibitor of bFGF) was used to determine the relationship between bFGF and putative EA-mediated improvements in synaptic plasticity. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed to evaluate hyperalgesia in rats with SNL. Tissue morphology was detected by hematoxylin-eosin (HE) and Nissl staining, while neural plasticity and its molecular mechanisms were examined by Western blotting, quantitative real-time polymerase chain reaction (qPCR), dual-label immunohistochemistry and transmission electron microscopy. RESULTS: We found that EA improved synaptic plasticity, consistent with higher levels of expression of bFGF and VEGF. Contrary to the beneficial effects of EA, NSC37204 promoted synaptic reconstruction. Furthermore, EA-induced improvements in the neurobehavioral state and improved synaptic plasticity were blocked by NSC37204, consistent with lower expression levels of bFGF and VEGF. CONCLUSION: These findings indicate that EA suppresses SNL-induced neuropathic pain by improving synaptic plasticity via upregulation of bFGF expression.

[Effect of electroacupuncture on angiopoietin-1 in spinal cord of rats with neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the behavior, histomorphology and the expression of angiopoietin-1 (Angpt-1) in rats with spinal nerve injury, so as to explore its mechanism on neuropathic pain. METHODS: Forty-five male SD rats were randomly divided into sham, model and EA groups (n=15 rats in each group). Spinal nerve ligation (SNL) of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the EA group were given EA at "Zusanli" (ST36) and "Kunlun" (BL60) of the operation side with continuous wave at a frequency of 2 Hz and an intensity of 1.5 mA once a day, 30 minutes each time for 7 days. The sham group only exposed L5 spinal nerves without ligation. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 3,5,7,10,12 and 14 after modeling. L4-L6 segments of spinal cord were taken and the morphological changes of spinal dorsal horn were observed by HE staining. The changes of spinal dorsal horn nerve fiber structure were observed by silver plating staining. Angpt-1 expression was detected by Western blot and immunohistochemistry. RESULTS: Compared with the sham group, the model group had significant reductions in MWT and TWL at each time point (P<0.01); compared with the model group, the EA group had significant increases in MWT and TWL on days 10,12 and 14 after intervention (P<0.05, P<0.01). HE staining showed that in the model group, the spinal dorsal horn showed degeneration and necrosis of neurons, nuclear fixation and shrinkage, and loose surrounding tissues. The degree of tissue damage of the EA group was milder than that of the model group. The silver staining results showed the model group had obvious neuronal fibrillary tangles, while there were fewer neuronal fibrillary tangles in the EA group. Compared with the sham group, the Angpt-1 expression in the model group was significantly decreased (P<0.01), and compared with the model group, the EA group had a significant increase in the expression of Angpt-1 (P<0.01). CONCLUSION: EA can promote the recovery of nerve function in SNL rats by up-regulating Angpt-1 expression.

[Influence of electroacupuncture on the expression of AMPA receptor subunit GluR1 in the spinal injured area of the rats with acute spinal cord injury].

OBJECTIVE: To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI. METHODS: A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T10. In the DNQX group, the intrathecal injection of 10 µL DNQX solution with a concentration of 1 nmol/µL was administered in 0.5 h after modeling success. In the EA group, EA (disperse-dense wave, 2 Hz/100 Hz in frequency, 0.5 mA in output current) was given at "Dazhui" (GV 14) and "Mingmen" (GV 4) in 0.5 h, 12 h and 24 h after modeling success for 30 min and totally 3 times. In the DNQX + EA group, the interventions in the above two groups were managed. The Basso, Beattie and Bresnahan locomotor rating score (BBB) was applied to evaluate the changes of locomotor function in the rats before modeling and in 6 h, 24 h and 48 h after modeling successively. Using the hematoxylin-eosin (HE) staining, the histopathological changes in the spinal anterior horn were observed in the spinal injured area. The immunofluorescence method was adopted to determine the number of GluR1 positive neuron of the spinal anterior horn. The Western blot method was used to determine the protein expression of GluR1 in the injured area. RESULTS: Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group (P<0.001) at the corresponding points. The BBB score was increased in each of intervention groups, but without statistical difference as compared with the model group (P>0.05). In the model group, it was found that the boundary between gray matter and white matter in the spinal anterior horn was blurred, the interstitial space enlarged, the neuron volume obviously shrunken, the cytoplasm decreased, the red stain deepened and some neuron nuclei fixed and shrunk. In the EA group, the morphology of the spinal anterior horn in the injured area was improved obviously, which was similar in the DNQX group and the DNQX + EA group. Compared with the sham-operation group, the GluR1 protein expression in the spinal injury area was increased (P<0.001) and the number of GluR1 positive neurons elevated (P<0.001) in the spinal anterior horn in the model group. Compared with the model group, in the EA group, the DNQX group and the DNQX + EA group, GluR1 protein expression was decreased (P<0.05, P<0.01) and the number of GluR1 positive neurons in the spinal anterior horn reduced (P<0.001). CONCLUSION: The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats.

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.

Electroacupuncture effects on the P2X4R pathway in microglia regulating the excitability of neurons in the substantia gelatinosa region of rats with spinal nerve ligation.

Electroacupuncture (EA) has been used to treat neuropathic pain induced by peripheral nerve injury (PNI) by applying an electrical current to acupoints with acupuncture needles. However, the mechanisms by which EA treats pain remain indistinct. High P2X4 receptor (P2X4R) expression levels demonstrate a notable increase in hyperactive microglia in the ipsilateral spinal dorsal horn following PNI. In order to demonstrate the possibility that EA analgesia is mediated in part by P2X4R in hyperactive microglia, the present study performed mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests in male Sprague­Dawley rats that had undergone spinal nerve ligation (SNL). The expression levels of spinal P2X4R were determined using reverse transcription­quantitative PCR, western blotting analysis and immunofluorescence staining. Furthermore, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded using whole­cell patch clamp to demonstrate the effect of EA on synaptic transmission in rat spinal substantia gelatinosa (SG) neurons. The results of the present study demonstrated that EA increased the MWT and TWL and decreased overexpression of P2X4R in hyperactive microglia in SNL rats. Moreover, EA attenuated the frequency of sEPSCs in SG neurons in SNL rats. The results of the present study indicate that EA may mediate P2X4R in hyperactive spinal microglia to inhibit nociceptive transmission of SG neurons, thus relieving pain in SNL rats.

Effects of electroacupuncture at different acupoints on the histomorphology of neurogenic bladder and the expression of hyperpolarization-activated cyclic nucleotide-gated channels in interstitial cells of Cajal in a rat model of suprasacral spinal cord injury.

BACKGROUND: To investigate the effects of electroacupuncture at different acupoints on the histomorphology of neurogenic bladder and the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in a rat model of suprasacral spinal cord injury (SCI). METHODS: A incomplete suprasacral SCI rat model was induced using a MASCIS impactor. Rats were randomly divided into a sham operation group, SCI model group, Ciliao treatment group or Guanyuan treatment group. The histomorphology of bladder cells was observed after hematoxylin and eosin (H&E) staining of bladder tissue sections. The expression of HCN channel proteins in ICC cells was detected by western blot and immunofluorescence, and HCN channel mRNA expression was measured using real-time PCR. RESULTS: In terms of histomorphology, the level of bladder cells after SCI increased significantly, and marked inflammation and edema were observed. Electroacupuncture treatment at the Ciliao acupoint significantly reduced inflammation and edema, whilst electroacupuncture treatment at the Guanyuan point partially reduced inflammation and edema. In terms of HCN channel protein and mRNA expression, western blotting, immunofluorescence and real-time PCR all confirmed that HCN channel expression after SCI was significantly upregulated, while electroacupuncture treatment at the Ciliao and Guanyuan acupoints inhibited HCN channel expression. CONCLUSIONS: Electroacupuncture treatment at the Ciliao acupoint significantly reduced histomorphological abnormalities in ICCs, and inhibited the expression of HCN channel proteins after SCI.

[Effect of electroacupuncture on the morphological changes of the spinal dorsal horn and the expression of p38 mitogen-activated protein kinase in the injured spinal cord of rats with neuropathic pain].

OBJECTIVE: To investigate the effect of electroacupuncture stimulation at "Zusanli"(ST36) and "Kunlun"(BL60) on the morphological changes of the spinal dorsal horn and the expression of p38 mitogen-activated protein kinase (p38MAPK) in the injured spinal cord of rats with neuropathic pain. METHODS: Male Sprague-Dawley rats were randomly divided into sham model group, model group, electroacupuncture group, and medication group, with 10 rats in each group. Spinal nerve ligation of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the electroacupuncture group were given electroacupuncture at ST36 and BL60 of the operation side with dilatational wave at a frequency of 2 Hz/100 Hz and an intensity of 1.5 mA once a day, 30 minutes each time, and those in the medication group were given intraperitoneal injection of 100 mg/mL Gabapentin solution (100 mg/kg) once a day; the one-week intervention was started at one week after surgery. Mechanical withdraw threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 1,3,5,7,10,12 and 14 after modeling, and the motor function of the affected hindlimb was scored. Methenamine silver stain was used to observe the morphological changes of the spinal dorsal horn, and Western blot was used to measure the relative protein expression of p38MAPK and phospho-p38MAPK(p-p38MAPK) in L4-L6 spinal segments. RESULTS: Compared with the sham model group, the model group had significant reductions in MWT and TWL at each time point (P<0.001) and a significant increase in motor function score (P<0.001); compared with the model group, the electroacupuncture group and the medication group had significant increases in MWT and TWL and a significant reduction in motor function score after treatment (P<0.05). The model group had obvious neuronal fibrillary tangles, particle vacuolar degeneration, and vacuoles containing argyrophilic grains in the cytoplasm of neural cells under a light microscope, while there were fewer neuronal fibrillary tangles in the electroacupuncture group and reduced vacuolar degeneration in the medication group. Compared with the sham model group, the model group had significant increases in the protein expression of p-p38MAPK in the spinal dorsal horn (P<0.001), and compared with the model group, the electroacupuncture group and the medication group had significant reductions in the protein expression of p-p38MAPK in the spinal dorsal horn(P<0.05). CONCLUSION: Electroacupuncture stimulation at ST36 and BL60 can increase pain threshold, improve the motor function of the affected hindlimb, and improve the necrosis of neurofibrils in the spinal dorsal horn in rats with neuropathic pain, possibly by regulating the expression of p-p38MAPK in the spinal dorsal horn.

Effect of electroacupuncture on the expression of P2 × 4, GABAA γ 2 and long-term potentiation in spinal cord of rats with neuropathic pain.

OBJECTIVE: To observe the impacts of electroacupuncture (EA) stimulation at "Zusanli and Kunlun Points" on spinal dorsal horn microglia activation in L5 spinal nerve ligation (SNL) rats and BNDF, P2 × 4 and GABAAγ2, and the changes in spinal dorsal horn synaptic plasticity in model rats. METHODS: Adult male SD rats (180-220 g) were selected and randomly divided into 6 groups, including the sham group, the SNL group, the SNL + EA group, the SNL+5-BDBD group, the SNL + EA + 5-BDBD group and the SNL + FEA group. The changes in the Iba-1, BDNF, P2 × 4 and GABAAγ2 in the spinal cord of rats were observed by Western blotting, immunofluorescence, RT-PCR and other techniques; the long-term changes in the potential after the excitatory synapse of the spinal dorsal horn in rats were observed by in vivo electrophysiological technique. RESULTS: After 7 days of intervention, the fluorescence intensity (FI) of P2 × 4 and Iba-1 in the SNL + EA group was lower than that in the SNL group and higher than that in the sham group(P < 0.01), but the FI of GABAAγ2 was higher than that in the SNL group(P < 0.01); the expression of Iba-1, BDNF and P2 × 4 proteins in the SNL + EA group, the SNL+5-BDBD group and the SNL + EA + 5-BDBD group was significantly lower than that in the SNL + FEA group(P < 0.05), but the expression of GABAAγ2 protein was higher (P < 0.05); after treatment with EA, the expression levels of Iba-1 mRNA and P2 × 4 mRNA in the SNL + EA group were lower than those in the SNL group(P < 0.01), but the expression levels of GABAAγ2 mRNA were higher (P < 0.01). Meanwhile, long-term potentiation changes could not be induced in the SNL + EA group. CONCLUSION: The EA stimulation at "Zusanli" and "Kunlun" points can improve the pain threshold of rats with neuropathic pain (NP), inhibit the excitatory postsynaptic potential (EPSP), and weaken the excitatory transmission efficiency between synapses during NP.

Hyperbaric oxygen therapy reduces apoptosis and dendritic/synaptic degeneration via the BDNF/TrkB signaling pathways in SCI rats.

Spinal cord injury (SCI) is a serious neurological disease without efficacious drugs. Anti-apoptosis and suppressing dendritic/synaptic degeneration in the anterior horn are essential targets after SCI. Previous studies found that hyperbaric oxygen therapy (HBOT) significantly protected rats after SCI. However, its potential effects and mechanisms remain unknown. The BDNF/TrkB signaling pathways evidently contribute to the SCI recovery. Currently, we mainly investigate the potential effects and mechanism of HBOT on anti-apoptosis and ameliorating impaired dendrites, dendritic spines and synapses after SCI. Establish SCI model and randomly divide rats into 5 groups. After SCI, rats were subjected to HBOT. ANA-12 is the specific inhibitor of BDNF/TrkB signal pathway. Changes in neurological deficit, neuronal morphology, apoptosis, protein expression and dendrite/synapse were examined by Basso-Beattie-Bresnahan (BBB) locomotor rating scale, Hematoxylin-eosin (HE) and Nissl staining, TUNEL staining, RT-PCR, Western blot, immunofluorescence and Golgi-Cox staining. We found HBOT suppressed dendritic/synaptic degeneration and alleviated apoptosis, consistent with the increase of BDNF and TrkB expression and improved neurological recovery. In contrast to the positive effects of HBOT, inhibitor increased degeneration and apoptosis. Moreover, we observed that these HBOT-mediated protective effects were significantly inhibited by inhibitor, consistent with the lower expression of BDNF/TrkB and worse neurobehavioral state. These findings suggest that hyperbaric oxygen therapy ameliorates spinal cord injury-induced neurological impairment by anti-apoptosis and suppressing dendritic/synaptic degeneration via upregulating the BDNF/TrkB signaling pathways.

[Influence of electroacupuncture of "Dazhui" (EX-B2) and "Mingmen" (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury].

OBJECTIVE: To observe the influence of eletroacupuncture (EA) at "Dazhui" (EX-B2) and "Mingmen" (GV4) on expression of NR2B subunit of N-methyl-D-aspartate receptor (NMDA) in the injured anterior horn (AH) area of rats with acute spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of neural repair. METHODS: A total of 96 male Sprague-Dawley (SD) rats were randomly and equally divided into four groups: sham operation (sham), model, medication (Methyl-prednisone, MP) and EA (n＝24 in each group). The acute SCI model was established by using a MASCIS spinal cord impactor. EA (2 Hz, 0.5 mA) was applied to EX-B2 and GV4 for 30 min, once at 0.5 h, 12 and 24 h after SCI. Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min (impact therapy) and 5.4 mg•kg－1•h－1 (maintaining treatment) 45 min thereafter for 23 h. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function 6, 24 and 48 h after SCI. Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin (H.E.) staining, and the expression levels of NR2B mRNA and protein of AH were measured by quantitative real-time PCR, Western blot and immunofluorescence, respectively. RESULTS: After SCI, the BBB scores at 6, 24 and 48 h were significantly decreased in the model group compared with those of the sham group (P<0.001), but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group (P>0.05). After modeling, the histopathological changes (blurred border of the grey-white matter, cellular karyopyknosis, deepening of the cytoplasmic red stain, and rupture, dissolution and disordered arrangement of myelinated nerve fibers) in the injury area of the spinal cord in the model group were apparent, the number of NR2B positive neurons and the relative expression levels of NR2B mRNA and protein were significantly increased in the model group relevant to the sham group (P<0.05). In contrast to the model group, the injured severity of the spinal cord AH region was relatively milder, and the expression levels of NR2B mRNA and protein were considerably down-regulated in both EA and MP groups (P<0.05). However, there were no significant differences between the EA and MP groups in the expression levels of NR2B mRNA and protein (P>0.05). CONCLUSION: EA at EX-B2 and GV4 may inhibit the expression of NR2B mRNA and protein in acute SCI rats, which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.

Atractylenolide III ameliorates cerebral ischemic injury and neuroinflammation associated with inhibiting JAK2/STAT3/Drp1-dependent mitochondrial fission in microglia.

BACKGROUND: Inflammation is a major contributor to stroke pathology, making it a promising strategy for intervention. Microglia, the resident macrophages in the brain, play essential roles in both the generation and resolution of neuroinflammation. In particular, mitochondrial homeostasis is critical for microglial function and its dysregulation is involved in the pathogenesis of ischemic stroke. Atractylenolide III (A III), a sesquiterpene lactone found in Atractylodes macrocephala Koidz, has been shown to have an inhibitory effect on inflammation. However, its effect specifically on neuroinflammation and microglial mitochondrial homeostasis following stroke remains elusive. HYPOTHESIS: We hypothesized that A III protects against brain ischemia through inhibition of neuroinflammation mediated by JAK2/STAT3/Drp1-dependent mitochondrial fission. METHODS: The neuroprotective and anti-neuroinflammatory effects of A III were investigated in vivo in mice with transient occlusion to the middle cerebral artery (MCAO) and in vitro in oxygen glucose deprivation-reoxygenation (OGDR)-stimulated primary microglia from mice. RESULTS: A III and AG490, an inhibitor of JAK2, treatment reduced brain infarct size, restored cerebral blood flow (CBF), ameliorated brain edema and improved neurological deficits in MCAO mice. Furthermore, A III and AG490 inhibited mRNA and protein expressions of proinflammatory (IL-1ß, TNF-α, and IL-6) and anti-inflammatory cytokines in both MCAO mice and OGDR-stimulated primary microglia. The JAK2/STAT3 pathway was effectively suppressed by A III, similar to the effect of AG490 treatment. In addition, A III and AG490 treatments significantly decreased Drp1 phosphorylation, translocation and mitochondrial fission in primary microglia stimulated with OGDR for 24 h. CONCLUSION: Our study demonstrated that A III was able to reduce complications associated with ischemia through inhibiting neuroinflammation, which was mediated in part by JAK2/STAT3-dependent mitochondrial fission in microglia.

Effect of electro-acupuncture on the BDNF-TrkB pathway in the spinal cord of CCI rats.

Microglia, which comprise a sensor for pathological events in the central nervous system, may be triggered by nerve injury and transformed from a quiescent state into an activated state; ionised calcium binding adaptor molecule 1 (Iba1) is a sensitive marker associated with activated microglia. Accumulated evidence suggests that spinal activated microglia and the brain-derived neurotrophic factor (BDNF)-tyrosine kinase receptor B (TrkB) signalling pathway play major roles in the production and development of neuropathic pain. Electro-acupuncture (EA) has a positive effect on relieving chronic neuropathic pain; however, the underlying mechanisms remain unclear. To determine the significance of EA in the treatment of neuropathic pain mediated by activated microglia and the BDNF-TrkB signalling pathway in the spinal cord, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) values were recorded to assess hyperalgesia and allodynia. In addition, the amount of activated microglia and BDNF were assessed via immunofluorescence. Iba1, BDNF and TrkB mRNA expression levels were examined using qPCR; the protein levels of BDNF, p-TrkB and TrkB in the spinal cord were analysed via western blotting. The present study demonstrated that EA treatment increased the MWT and TWL values. EA significantly inhibited the proportion of activated microglia and BDNF expression in the spinal cord after chronic constrictive injury (CCI). Furthermore, EA decreased the expression of BDNF and TrkB at both the mRNA and protein levels in the spinal cord of CCI rats. These findings suggest that the analgesic effect of EA may be achieved by inhibiting the activation of spinal microglia and subsequently blocking the BDNF-TrkB signalling pathway.

KCC2-GABAA pathway correlates with the analgesic effect of electro-acupuncture in CCI rats.

Potassium-chloride cotransporter 2 (KCC2) has been indicated to serve a crucial role during chronic neuropathic pain (NP). Following the emergence of NP, γ­aminobutyric acid (GABA) A receptor­mediated signaling may be further impaired by the changes of KCC2 chloride anion gradient. In the present study, the authors investigate the effect of electro-acupuncture (EA) on the behavior and the expression of KCC2 and GABAA receptor γ2 subunit in the spinal cord of chronic constriction injury (CCI) model rats. A total of 60 adult male Sprague­Dawley rats were divided into four groups: Normal group, sham­CCI group, CCI group and CCI+EA group. The effect of EA was assessed via the values of mechanical withdrawal threshold and thermal withdrawal latency, which were significantly improved upon stimulation of the ST­36 and GB­34 acupoints. In addition, a marked reduction in both the mRNA and protein levels of KCC2 and GABAA receptor γ2 subunit was observed in the spinal cord following loose ligation of the sciatic nerve. The reductions in KCC2 and GABAA receptor γ2 subunit expression were reversed by EA treatment. These results support the notion that KCC2 and GABAA receptor γ2 subunit contribute to NP following peripheral nerve injury and extend the understanding of the analgesic effects of EA on NP.

[Effects of electroacupuncture on activation of microglia cells in spinal cord in rats with neuropathic pain].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the activation of microglia cells in the L4 to L6 spinal cord in rats with neuropathic pain, so as to investigate whether EA could inhibit the activation of spinal microglial cells and regulate the expression of brain-derived neurotrophic factor (BDNF) to achieve the analgesic effect. METHODS: Forty male Sprague Dawley rats were randomly divided into a normal group, a sham-model group, a model group and an EA group, 10 rats in each one. The rats in the normal group received no treatment; the rats in sham-model group were treated with operation to exposure sciatic nerve for 2 to 3 min (no knot); the rats in the remaining groups were treated with model establishment of chronic constrictive injury (CCI). 7 days after model establishment, the rats in the EA group were treated with EA at "Zusanli" (ST 36) and "Yanglingquan" (GB 34), 30 min per time, once a day for consecutive 7 days. Only immobilization was used in the remaining groups the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of affected side feet were respectively measured before model establishment and 3 days, 5 days, 7 days, 10 days, 12 days and 14 days after model establishment; 14 days after model establishment, rats were sacrificed; the immunohistochemical method was used to measure the expression of Iba1 and BDNF in the sample of L4 to L6 spinal cord; real-time fluorescent quantitative PCR was used to measure the expression BDNF mRNA. RESULTS: Compared with the sham-model group, the pain threshold was decreased significantly in the model group (P<0.05), leading to hyperpathia. After EA treatment, compared with the model group, the pain threshold was increased significantly in the EA group (P<0.05). 14 days after operation, the microglia cells in the L4 to L6 spinal cord, expression of BDNF and level of mRNA in the model group were significantly higher than those in the normal group and sham-model group (all P<0.01); those in the EA group were significantly lower than those in the model group (all P<0.01). CONCLUSIONS: The analgesic effect on neuropathic pain is likely to be achieved by EA through inhibiting the activation of spinal microglia cells and down-regulating the expression of BDNF.

Electro-acupuncture at Governor Vessel improves neurological function in rats with spinal cord injury.

OBJECTIVE: To determine the effects of electro-acupuncture (EA) at Governor Vessel (GV) on the locomotor function in spinal cord injury (SCI) rats and explore the underlying mechanism. METHODS: Thirtytwo male Sprague-Dawley rats were randomly divided into four groups namely: the sham group (with sham operation); the untreated group (without treatment after spinal cord impact); the EA-1 group [EA applied at Baihui (GV 20) and Fengfu (GV 16) after spinal cord impact] and the EA-2 group [with EA applied at Dazhui (GV 14) and Mingmen (GV 4) after spinal cord impact]. Real-time quantitative-polymerase chain reaction (RT-PCR) and Western Blotting were used to assess changes in the mRNA and protein expression levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) at 7 weeks following EA administration. In addition, the Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale was assessed at 1 day, 1 week, 3 weeks and 7 weeks post-injury. RESULTS: The results showed that EA stimulation induced neuroprotective effects after SCI correlated with the up-regulation of BDNF and NT-3 (P<0.05). Furthermore, EA stimulation at GV 14 and GV 4 could significantly promote the recovery of locomotor function and this may be linked to the up-regulation of BDNF and NT-3 (P<0.05). CONCLUSIONS: EA treatment applied at GV acupoints either within the injury site or adjacent undamaged regions near the brain can improve functional recovery, which may be correlated with the upregulation of BDNF and NT-3. In addition, it would be more effective to administer EA at GV 14 and GV 4 near the injury site of the SCI rats.