A neural circuit associated with anxiety-like behaviors induced by chronic inflammatory pain and the anxiolytic effects of electroacupuncture.

AIMS: Negative emotions induced by chronic pain are a serious clinical problem. Electroacupuncture (EA) is a clinically proven safe and effective method to manage pain-related negative emotions. However, the circuit mechanisms underlying the effect of EA treatment on negative emotions remain unclear. METHODS: Plantar injection of complete Freund's adjuvant (CFA) was performed to establish a rat model of chronic inflammatory pain-induced anxiety-like behaviors. Adeno-associated virus (AAV) tracing was used to identify excitatory synaptic transmission from the rostral anterior cingulate cortex (rACC) to the dorsal raphe nucleus (DRN). Employing chemogenetic approaches, we examined the role of the rACC-DRN circuit in chronic pain-induced anxiety-like behaviors and investigated whether EA could reverse chronic pain-induced dysfunctions of the rACC-DRN circuit and anxiety-like behaviors. RESULTS: We found that chemogenetic activation of the rACC-DRN circuit alleviated CFA-induced anxiety-like behaviors, while chemogenetic inhibition of the rACC-DRN circuit resulted in short-term CFA-induced anxiety-like behaviors. Further research revealed that the development of CFA-induced anxiety-like behaviors was attributed to the dysfunction of rACC CaMKII neurons projecting to DRN serotonergic neurons (rACCCaMKII-DRN5-HT neurons) but not rACC CaMKII neurons projecting to DRN GABAergic neurons (rACCCaMKII-DRNGABA neurons). This is supported by the findings that chemogenetic activation of the rACCCaMKII-DRN5-HT circuit alleviates anxiety-like behaviors in rats with chronic pain, whereas neither chemogenetic inhibition nor chemogenetic activation of the rACCCaMKII-DRNGABA circuit altered CFA chronic pain-evoked anxiety-like behaviors in rats. More importantly, we found that EA could reverse chronic pain-induced changes in the activity of rACC CaMKII neurons and DRN 5-HTergic neurons and that chemogenetic inhibition of the rACCCaMKII-DRN5-HT circuit blocked the therapeutic effects of EA on chronic pain-induced anxiety-like behaviors. CONCLUSIONS: Our data suggest that the reversal of rACCCaMKII-DRN5-HT circuit dysfunction may be a mechanism underlying the therapeutic effect of EA on chronic pain-induced anxiety-like behaviors.

Electroacupuncture alleviates mechanical allodynia and anxiety-like behaviors induced by chronic neuropathic pain via regulating rostral anterior cingulate cortex-dorsal raphe nucleus neural circuit.

AIMS: Epidemiological studies in patients with neuropathic pain have demonstrated a strong association between neuropathic pain and psychiatric conditions such as anxiety. Preclinical and clinical work has demonstrated that electroacupuncture (EA) effectively alleviates anxiety-like behaviors induced by chronic neuropathic pain. In this study, a potential neural circuitry underlying the therapeutic action of EA was investigated. METHODS: The effects of EA stimulation on mechanical allodynia and anxiety-like behaviors in animal models of spared nerve injury (SNI) were examined. EA plus chemogenetic manipulation of glutamatergic (Glu) neurons projecting from the rostral anterior cingulate cortex (rACCGlu ) to the dorsal raphe nucleus (DRN) was used to explore the changes of mechanical allodynia and anxiety-like behaviors in SNI mice. RESULTS: Electroacupuncture significantly alleviated both mechanical allodynia and anxiety-like behaviors with increased activities of glutamatergic neurons in the rACC and serotoninergic neurons in the DRN. Chemogenetic activation of the rACCGlu -DRN projections attenuated both mechanical allodynia and anxiety-like behaviors in mice at day 14 after SNI. Chemogenetic inhibition of the rACCGlu -DRN pathway did not induce mechanical allodynia and anxiety-like behaviors under physiological conditions, but inhibiting this pathway produced anxiety-like behaviors in mice at day 7 after SNI; this effect was reversed by EA. EA plus activation of the rACCGlu -DRN circuit did not produce a synergistic effect on mechanical allodynia and anxiety-like behaviors. The analgesic and anxiolytic effects of EA could be blocked by inhibiting the rACCGlu -DRN pathway. CONCLUSIONS: The role of rACCGlu -DRN circuit may be different during the progression of chronic neuropathic pain and these changes may be related to the serotoninergic neurons in the DRN. These findings describe a novel rACCGlu -DRN pathway through which EA exerts analgesic and anxiolytic effects in SNI mice exhibiting anxiety-like behaviors.

Electroacupuncture inhibits the interaction between peripheral TRPV1 and P2X3 in rats with different pathological pain.

Chronic pain is regarded to be one of the common and refractory diseases to cure in the clinic. One hundred Hz electroacupuncture (EA) is commonly used for inflammatory pain and 2 Hz for neuropathic pain possibly by modulating the transient receptor potential vanilloid subtype 1 (TRPV1) or the purinergic P2X3 related pathways. To clarify the mechanism of EA under various conditions of pathological pain, rats received a subcutaneous administration of complete Freund's adjuvant (CFA) for inflammatory pain and spared nerve injury (SNI) for neuropathic pain. The EA was performed at the bilateral ST36 and BL60 1 d after CFA or SNI being successfully established for 3 consecutive days. The mechanical hyperalgesia test was measured at baseline, 1 d after model establishment, 1 d and 3 d after EA. The co-expression changes, co-immunoprecipitation of TRPV1 and P2X3, and spontaneous pain behaviors (SPB) test were performed 3 d after EA stimulation. One hundred Hz EA or 2Hz EA stimulation could effectively down-regulate the hyperalgesia of CFA or SNI rats. The increased co-expression ratio between TRPV1 and P2X3 at the dorsal root ganglion (DRG) in two types of pain could be reduced by 100Hz or 2Hz EA intervention. While 100Hz or 2Hz EA was not able to eliminate the direct physical interaction between TRPV1 and P2X3. Moreover, EA could significantly inhibit the SPB induced by the co-activation of peripheral TRPV1 and P2X3. All results indicated that EA could significantly reduce the hyperalgesia and the SPB, which was partly related to inhibiting the co-expression and indirect interaction between peripheral TRPV1 and P2X3.

Rostral Anterior Cingulate Cortex-Ventrolateral Periaqueductal Gray Circuit Underlies Electroacupuncture to Alleviate Hyperalgesia but Not Anxiety-Like Behaviors in Mice With Spared Nerve Injury.

Neuropathic pain is a common cause of chronic pain and is often accompanied by negative emotions, making it complex and difficult to treat. However, the neural circuit mechanisms underlying these symptoms remain unclear. Herein, we present a novel pathway associated with comorbid chronic pain and anxiety. Using chemogenetic methods, we found that activation of glutamatergic projections from the rostral anterior cingulate cortex (rACC Glu ) to the ventrolateral periaqueductal gray (vlPAG) induced both hyperalgesia and anxiety-like behaviors in sham mice. Inhibition of the rACC Glu -vlPAG pathway reduced anxiety-like behaviors and hyperalgesia in the spared nerve injury (SNI) mice model; moreover, electroacupuncture (EA) effectively alleviated these symptoms. Investigation of the related mechanisms revealed that the chemogenetic activation of the rACC Glu -vlPAG circuit effectively blocked the analgesic effect of EA in the SNI mice model but did not affect the chronic pain-induced negative emotions. This study revealed a novel pathway, the rACC Glu -vlPAG pathway, that mediates neuropathic pain and pain-induced anxiety.

Effect of Electroacupuncture on Pain Perception and Pain-Related Affection: Dissociation or Interaction Based on the Anterior Cingulate Cortex and S1.

Electroacupuncture (EA) can effectively modulate pain perception and pain-related negative affect; however, we do not know whether the effect of EA on sensation and affect is parallel, or dissociated, interactional. In this study, we observed the effects of the anterior cingulate cortex (ACC) lesion and the primary somatosensory cortex (S1) activation on pain perception, pain-related affection, and neural oscillation in S1. ACC lesions did not affect pain perception but relieved pain-paired aversion. S1 activation increased pain perception and anxious behavior. EA can mitigate pain perception regardless of whether there is an ACC lesion. Chronic pain may increase the delta and theta band oscillatory activity in the S1 brain region and decrease the oscillatory activity in the alpha, beta, and gamma bands. EA intervention may inhibit the oscillatory activity of the alpha and beta bands. These results suggest that EA may mitigate chronic pain by relieving pain perception and reducing pain-related affection through different mechanisms. This evidence builds upon findings from previous studies of chronic pain and EA treatment.

[Effect of electroacupuncture on expression of GABAA receptor mRNA in different brain regions in rats with chronic inflammatory pain].

OBJECTIVE: To observe the expression of GABAA receptor mRNA in different brain regions of the central nervous system in chronic inflammatory pain rats and the intervention effect of electroacupuncture (EA). METHODS: A total of 48 SPF male SD rats were randomly divided into a blank control group, a model control group, an EA group and a sham EA group, 12 rats in each group. The model of chronic inflammatory pain was established by injecting Freund's complete adjuvant into the foot. The EA group was treated with EA 28 days after the model establishment. The "Housanli" (ST 36) and "Kunlun" (BL 60) were selected and treated with dilatational wave, 2 Hz/100 Hz in frequency, 0.5-1.5 mA for 30 min; EA was given only once. In the sham EA group, the same acupoints were selected but the needles were only inserted into subcutaneous area; EA was connected for 30 min without electrical stimulation. The behavior changes of mechanical pain threshold and thermal pain threshold before model establishment, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after the model establishment as well as emotional behavior 29 days after the model establishment were observed; the relative expressions of GABAA receptor mRNA in anterior cingulate cortex, amygdala and hypothalamus were observed. RESULTS: Compared with the blank control group, the change rates of mechanical pain threshold and thermal pain threshold in the model control group were decreased significantly 1 day, 3 days, 7 days, 14 days, 21 days, 28 days after model establishment (P<0.01); 29 days after model establishment, the movement distance and staying time in the central area of open field test in the model control group were decreased significantly (P<0.05). After EA intervention, compared with the model control group and the sham EA group, the change rates of mechanical pain threshold and thermal pain threshold, as well as the movement distance and the staying time of central area were significantly increased in the EA group (P<0.01, P<0.05). Twenty-nine days after model establishment, the expression of GABAA receptor mRNA in anterior cingulate cortex and hypothalamus was not significantly different among all groups (P>0.05). Compared with the blank control group, the expression of GABAA receptor mRNA in the amygdala was decreased significantly in the model control group (P<0.01); compared with the model control group and the sham EA group, the expression of GABAA receptor mRNA in amygdala was increased after intervention in the EA group (P<0.01). CONCLUSION: Single treatment of EA could significantly increase the mechanical pain threshold and thermal pain threshold, improve abnormal emotional behavior in rats with chronic inflammatory pain, which may be related to the increasing of expression of GABAA receptor mRNA in the amygdala.

Electroacupuncture Alleviates Chronic Pain-Induced Anxiety Disorders by Regulating the rACC-Thalamus Circuitry.

Anxiety is a common comorbidity associated with chronic pain, which results in chronic pain complexification and difficulty in treatment. Electroacupuncture (EA) is commonly used to treat chronic pain and anxiety. However, the underlying mechanisms of the EA effect are largely unknown. Here, we showed that a circuitry underlying chronic pain induces anxiety disorders, and EA can treat them by regulating such circuitry. Using chemogenetic methods, we found that chemogenetic activation of the rostral anterior cingulate cortex (rACC) glutamatergic output to the thalamus induced anxiety disorders in control rats. Then, chemogenetic inhibition of the rACC-thalamus circuitry reduced anxiety-like behavior produced by intraplantar injection of the complete Freund's adjuvant (CFA). In this study, we examined the effects of EA on a rat model of CFA-mediated anxiety-like behaviors and the related mechanisms. We found that chemogenetic activation of the rACC-thalamus circuitry effectively blocked the effects of EA on chronic pain-induced anxiety-like behaviors in CFA rats. These results demonstrate an underlying rACC-thalamus glutamatergic circuitry that regulates CFA-mediated anxiety-like behaviors. This study also provides a potential mechanistic explanation for EA treatment of anxiety caused by chronic pain.

Electroacupuncture Stimulation Alleviates CFA-Induced Inflammatory Pain Via Suppressing P2X3 Expression.

Chronic inflammatory pain is one of the most common complaints that seriously affects patients' quality of life. Previous studies have demonstrated that the analgesic effect of electroacupuncture (EA) stimulation on inflammatory pain is related to its frequency. In this study, we focused on whether the analgesic effects of EA are related to the period of stimulation. Purinergic receptor P2X3 (P2X3) is involved in the pathological process underlying chronic inflammatory pain and neuropathic pain. We hypothesized that 100 Hz EA stimulation alleviated Freund's complete adjuvant (CFA) induced inflammatory pain via regulating P2X3 expression in the dorsal root ganglion (DRG) and/or spinal cord dorsal horn (SCDH). We also assumed that the analgesic effect of EA might be related to the period of stimulation. We found that both short-term (three day) and long-term (14 day) 100 Hz EA stimulation effectively increased the paw withdrawal threshold (PWT) and reversed the elevation of P2X3 in the DRG and SCDH of CFA rats. However, the analgesic effects of 100 Hz EA were not dependent on the period of stimulation. Moreover, P2X3 inhibition or activation may contribute to or attenuate the analgesic effects of 100 Hz EA on CFA-induced inflammatory pain. This result indicated that EA reduced pain hypersensitivity through P2X3 modulation.

Effect of Electroacupuncture in "Zusanli" and "Kunlun" Acupoints on TLR4 Signaling Pathway of Adjuvant Arthritis Rats.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Previous study suggested that toll-like receptor (TLR) signaling pathway contributes to the development and progression of RA. In recent years, acupuncture has become one of the most vital treatments of arthralgia. But little is known about the mechanisms of improving RA by acupuncture. STUDY QUESTION: The study studied the effect of electroacupuncture in "Zusanli" and "Kunlun" acupoints on the expression of TLR4, myeloid differentiation factor 88 (MYD88), and NF-κB in adjuvant arthritis rats to clarify the molecular mechanism of acupuncture of RA. STUDY DESIGN: A rat model of adjuvant arthritis was established with injection of 0.1 mL Freund complete adjuvant in the right hindlimb footpad. We next punctured the Zusanli and Kunlun acupoints with 0.25 × 40-mm acupuncture needles to 5-mm depth. Then, we performed electroacupuncture treatment for 28 days with frequency of 2 Hz and intensity of 2 mA, once a day and 30 minutes each time. MEASURES AND OUTCOMES: Arthritis index and paw swelling were measured every week. FQ-PCR and western blot were used to detect the expression of TLR4, MYD88, and NF-κB. RESULTS: Paw swelling of rats injected with Freund complete adjuvant was more serious than that of the normal rats, which illustrated the successful establishment of adjuvant arthritis rat model. After treatment for 14 days, the paw swelling and joint symptoms score decreased, paw tissue inflammation eased in the rats of treatment group compared with the model group during the same period. After treatment for 28 days, the expression of TLR4, MYD88, and NF-κB in the ankle bone tissues decreased at both mRNA and protein levels. CONCLUSIONS: Stimulation with electric needle in Zusanli and Kunlun acupoints can reduce the expression of TLR4, MYD88, and NF-κB, which play an important role in treatment of adjuvant arthritis.

Tanshinone IIA Sodium sulfonate regulates antioxidant system, inflammation, and endothelial dysfunction in atherosclerosis by downregulation of CLIC1.

BACKGROUND: Tanshinone IIA Sodium sulfonate (STS) is clinically used for treating cardiovascular diseases in Traditional Chinese Medicine due to its antioxidation and anti-inflammation activities. Intracellular chloride channel 1 (CLIC1) participates in the regulation of oxidative stress and inflammation. This study investigates whether CLIC1 mediates the cardioprotective effects of STS. METHODS: STS were used to treat atherosclerosis (AS) induced by feeding Apolipoprotein E-deficient (ApoE-/-) mice with a high-fat, cholesterol-rich diet. In addition, normal and CLIC1-/- human umbilical vein endothelial cells were treated with STS after exposure to H2O2 for 12h. The oxidative status was determined by analyzing reactive oxygen species(ROS) and malondialdehyde (MDA) levels. ELISA, qRT-PCR and Western blot were used to determine the levels of TNF-α, IL-6, ICAM-1 and VCAM-1. CLIC1 cellular localization was examined by immunofluorescence. Chloride ion concentration was detected with chloride ion quenchers (MQAE). RESULTS: STS treatment decreased atherosclerotic lesion area by 3.5 times (P = 0.001) in vivo. Meanwhile, STS reduced MDA production (13.6%, P = 0.008), increased SOD activity (113.6%, P = 0.008), decreased TNF-α (38.6%, P = 0.008) and IL-6 (43.0%, P = 0.03) levels, and downregulated the expression of CLIC1, ICAM-1, and VCAM-1 in the atherosclerotic mice. The dose-dependent anti-oxidative and anti-inflammatory effects of STS were further confirmed in vitro. Furthermore, CLIC1 depletion abolished the STS-mediated decrease of ROS and MDA production in HUVEC cells. Additionally, STS inhibited both CLIC1 membrane translocation and chloride ion concentration. CONCLUSION: The anti-oxidant, and anti-inflammation properties of STS in preventing AS is mediated by its inhibition of CLIC1 expression and membrane translocation.

Enantioselective Phytotoxicity and the Relative Mechanism of Current Chiral Herbicides.

BACKGROUND: Regardless of the achievable of chiral switch, most of the chiral nature agrochemical is still sold as racemate or enantiomer-enriched pesticides. Herbicides, accounted for a large proportion in pesticide market, are of great concern due to the frequent occurrence in environment and the structure selective phyto-biochemical impact on plants. METHODS: We give a systematic search on the literature database and included approximately 50 papers which were related to the review. We do careful categories for the chiral herbicides according to their structure and listed out the acute phytotoxicity endpoints. The potential mechanism for the enantioselective toxicity was concluded into 5 main points. RESULTS: The enantiomer-specific toxicity on plant growth and flowers are limited on phenoxyalkanoic acid herbicide, aryloxyphenoxypropanoic acid, imidazolinone herbicide, and acetamide pesticide. Data available on the potential mechanism explanation of enantioselective phytotoxicity has been concerned on the genetic transcription, oxidative stress, and photosynthesis disruption, etc. A comparison between the two enantiomers' enantioselective effects identified an organ-specific and species-specific phenomenon for several herbicides. Moreover, a more herbicidal activity enantiomer is also displayed the more toxicity than its antipode. CONCLUSION: The review elucidated a paucity of information on the enantioselective effect research on various types of plants at the different life stages. It appealed us to conduct a more holistic approach to balance the benefit between herbicidal activity and phytotoxicity when try to develop an enantio-pure herbicide.