Electroacupuncture Alleviates Hyperalgesia and Anxiety-Like Behaviors in Pain Memory Model Rats Through Activation of GABAergic Neurons and GABA Receptor in the Rostral Anterior Cingulate Cortex.

Recent studies have confirmed that pain memory is often accompanied by negative emotions. Electroacupuncture (EA) can block the retrieval of painful memories, thereby alleviating the associated negative behaviors. However, the underlying mechanism is poorly understood. This study revealed that the effect of EA on pain memory-induced negative behaviors is related to the mediation of GABAergic neuron activity and GABA receptor expression in the rostral anterior cingulate cortex (rACC). Previous studies have shown that the rACC is a crucial area for regulating nociceptive behaviors and negative emotions in pain memory models. The GABAergic neurons and receptors in the rACC are largely involved in pain sensation and related effects. However, the relationships among pain memory, GABAergic neurons and receptors in the rACC have not been investigated. In this study, we established a pain memory model via secondary plantar cross-injection of carrageenan and EA treatment. Using chemogenetic methods and behavioral assessments of pain and negative emotion, we found that early excitation of GABAergic neurons in the rACC blocked the recall of pain memories and reduced anxiety-like behaviors in pain memory model rats. Furthermore, pharmacological methods revealed that excitation of GABAA and GABAB receptors in the rACC blocks hyperpathia associated with pain memory and pain-induced anxiety-like behaviors, while inhibition of GABAA and GABAB receptors reverses these effects. These results suggest that EA may alleviate pain and associated anxiety-like behaviors related to pain memories through the activation of GABAergic neurons and excitation of GABAA and GABAB receptors in the rACC.

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 the relapse of pain-related aversive memory by activating KOR and inhibiting GABAergic neurons in the insular cortex.

Pain-related aversive memory is common in chronic pain patients. Electroacupuncture has been demonstrated to block pain-related aversive memory. The insular cortex is a key region closely related to aversive behaviors. In our study, a potential mechanism underlying the effect of electroacupuncture treatment on pain-related aversive memory behaviors relative to the insular cortex was investigated. Our study used the chemogenetic method, pharmacological method, electroacupuncture intervention, and behavioral detection. Our study showed that both inhibition of gamma-aminobutyric acidergic neurons and activation of the kappa opioid receptor in the insular cortex blocked the pain-related aversive memory behaviors induced by 2 crossover injections of carrageenan in mice; conversely, both the activation of gamma-aminobutyric acidergic neurons and inhibition of kappa opioid receptor in the insular cortex play similar roles in inducing pain-related aversive memory behaviors following 2 crossover injections of carrageenan. In addition, activation of gamma-aminobutyric acidergic neurons in the insular cortex reversed the effect of kappa opioid receptor activation in the insular cortex. Moreover, electroacupuncture effectively blocked pain-related aversive memory behaviors in model mice, which was reversed by both activation of gamma-aminobutyric acidergic neurons and inhibition of kappa opioid receptor in the insular cortex. The effect of electroacupuncture on blocking pain-related aversive memory behaviors may be related to the activation of the kappa opioid receptor and inhibition of gamma-aminobutyric acidergic neurons in the insular cortex.

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 Pain Memory and Related Anxiety-Like Behaviors by Blockading the GABAB Receptor Function in the Midcingulate Cortex.

Pain memory is commonly considered an underlying cause of chronic pain and is also responsible for a range of anxiety. Electroacupuncture (EA) has been shown to ameliorate pain memories and exert anti-anxiety effects. Previous research has indicated that GABAergic neurons and/or GABA receptors (GABARs) in the midcingulate cortex (MCC) have potential associations with chronic pain and anxiety. However, there is no known empirical research that has specifically studied the effects of EA on the GABAergic system in the MCC. Here, we used cross-injection of carrageenan to establish the pain memory rats model. Immunofluorescence were used to detect the excitability of GABAergic neurons within MCC. Von Frey filament, elevated zero maze, and open field tests were used to measure mechanical allodynia and anxiety-like behaviors, combined with chemogenetic and pharmacologic technologies. Finally, this study provides evidence that pain memories contribute to generalized negative emotions and that downregulating the activity of GABAergic neurons within MCC could block pain memories and reverse anxiety emotion. Specifically, GABABR is involved in pain memory and related anxiety-like behaviors. Activation of GABAergic neurons in the MCC did not reverse the effects of EA on pain memories and related anxiety-like behaviors, whereas these effects could be reversed by a GABABR agonist. These findings highlight the functional significance of GABABR in the EA-mediated attenuation of pain memories and related anxiety-like behaviors in rats.

Electroacupuncture Alleviates Anxiety-Like Behaviors Induced by Chronic Neuropathic Pain via Regulating Different Dopamine Receptors of the Basolateral Amygdala.

Chronic pain, such as neuropathic pain, causes anxiety and other negative emotions, which aggravates the pain sensation and increases the risk of chronic pain over time. Dopamine receptor D1 (DRD1) and dopamine receptor D2 (DRD2) in the basolateral amygdala (BLA) have been implicated in mediating anxiety-related behaviors, but their potential roles in the BLA in neuropathic pain-induced anxiety have not been examined. Electroacupuncture (EA) is commonly used to treat chronic pain and emotional disorders, but it is still unclear whether EA plays a role in analgesia and anxiety relief through DRD1 and DRD2 in the BLA. Here, we used western blotting to examine the expression of DRD1 and DRD2 and pharmacological regulation combined with behavioral testing to detect anxiety-like behaviors. We observed that injection of the DRD1 antagonist SCH23390 or the DRD2 agonist quinpirole into the BLA contributed to anxiety-like behaviors in naive mice. EA also activated DRD1 or inhibited DRD2 in the BLA to alleviate anxiety-like behaviors. To further demonstrate the role of DRD1 and DRD2 in the BLA in spared nerve injury (SNI) model-induced anxiety-like behaviors, we injected the DRD1 agonist SKF38393 or the DRD2 antagonist sulpiride into the BLA. We found that both activation of DRD1 and inhibition of DRD2 could alleviate SNI-induced anxiety-like behaviors, and EA had a similar effect of alleviating anxiety. Additionally, neither DRD1 nor DRD2 in the BLA affected SNI-induced mechanical allodynia, but EA did. Overall, our work provides new insights into the mechanisms of neuropathic pain-induced anxiety and a possible explanation for the effect of EA treatment on anxiety caused by chronic pain.

Electroacupuncture Reverses CUMS-Induced Depression-Like Behaviors and LTP Impairment in Hippocampus by Downregulating NR2B and CaMK II Expression.

OBJECTIVE: Depression is a global mental health problem with high disability rate, which brings a huge disease burden to the world. Electroacupuncture (EA) has been shown to be an effective method for the treatment of depression. However, the mechanism underling the antidepressant effect of EA has not been clearly clarified. The change of synaptic plasticity is the focus in the study of antidepressant mechanism. This study will observe the effect of EA on LTP of hippocampal synaptic plasticity and explore its possible mechanism. METHODS: The depression-like behavior rat model was established by chronic unpredictable mild stress (CUMS). EA stimulation (Hegu and Taichong) was used to treat the depressed rats. The depression-like behavior of rats was tested by weight measurement, open field test, depression preference test, and novelty suppressed feeding test. Long-term potentiation (LTP) was recorded at CA1 synapses in hippocampal slices by electrophysiological method. N-methyl-D-aspartate receptor subunit 2B (NR2B) and calmodulin-dependent protein kinase II (CaMK II) protein levels were examined by using western blot. RESULTS: After the establishment of CUMS-induced depression model, the weight gain rate, sucrose preference rate, line crossing number, and rearing times of rats decreased, and feeding time increased. At the same time, the LTP in hippocampus was impaired, and the expressions of NR2B and CaMK II were upregulated. After EA treatment, the depression-like behavior of rats was improved, the impairment of LTP was reversed, and the expression levels of NR2B and CaMK II protein were downregulated. CONCLUSION: EA can ameliorate depression-like behaviors by restoring LTP induction, downregulating NR2B and CaMK II expression in CUMS model rats, which might be part of the mechanism of EA antidepressant.

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.

[Comparison of the effect between electroacupuncture and NSAIDs on pain memory based on cAMP/PKA/CREB pathway in anterior cingulate gyrus].

OBJECTIVE: To observe the direct intervention effects of electroacupuncture (EA) and non-steroid anti-inflammatory drugs (NSAIDs) on pain memory, and to explore their effects on cAMP/PKA/cAMP pathway in anterior cingulate gyrus (ACC). METHODS: Fifty clean healthy male SD rats were randomly divided into a control group, a model group, an indomethacin group, an EA group and a sham EA group, 10 rats in each group. Except the control group, the pain memory model was established in the remaining four groups by twice injection of carrageenan at foot; 0.1 mL of 2%λ-carrageenan was subcutaneously injected at the left foot of rats; 14 days later, when the pain threshold of rats of each group returned to the basic level, the second injection was performed with the same procedure. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36) for 30 min; the rats in the indomethacin group was treated with indomethacin intragastric administration with the dose of 3 mg/kg; the rats in the sham EA group was treated with EA without electricity at the point 0.3 mm forward "Zusanli" (ST 36) with the depth of 2 mm for 30 min; the rats in the control group was not given any invention. All the above interventions were performed 5 h, 1 d, 2 d and 3 d after the second injection of 2% λ-carrageenan. The left-side paw withdrawal thresholds (PWT) were observed before the first injection, 4 h, 3 d, 5 d after the first injection, before the second injection and 4 h, 1 d, 2 d, 3 d after the second injection. Three days after the second injection, the number of positive cells of cAMP, p-PKA, p-CREB and the number of positive cells of protein co-expression in the right ACC brain area were detected by immunofluorescence, and the relative protein expression of p-PKA and p-CREB were detected by Western blot. RESULTS: Compared with the control group, the PWTs in the model group decreased significantly 4 h, 3 d and 5 d after the first injection and 1 d, 2 d and 3 d after the second injection (P<0.05); compared with the control group, the positive expression of cAMP, p-PKA and p-CREB in the right ACC brain area in the model group increased significantly (P<0.05), and the number of positive cells of the co-expression of cAMP/p-PKA and p-PKA/p-CREB also increased significantly (P<0.05). Compared with the model group, indomethacin group and sham EA group, the PWTs in the EA group were increased significantly 1 d, 2 d and 3 d after the second injection (P<0.05); compared with the model group, indomethacin group and sham EA group, the positive expression of p-PKA and p-CREB in the right ACC brain area in the EA group decreased significantly (P<0.05), and the number of positive cells of co-expression of cAMP/p-PKA and p-PKA/p-CREB was decreased significantly (P<0.05). Compared with the model group and sham EA group, the positive expression of cAMP in the right ACC brain area was decreased in the EA group (P<0.05). CONCLUSION: EA have a direct intervention effect on pain memory, which have significant advantage over NSAIDs in the treatment of chronic pain. The advantage effect of EA on pain memory may be related to the inhibition of cAMP/PKA/CREB pathway in ACC area.

Electroacupuncture Improved Chronic Cerebral Hypoperfusion-Induced Anxiety-Like Behavior and Memory Impairments in Spontaneously Hypertensive Rats by Downregulating the ACE/Ang II/AT1R Axis and Upregulating the ACE2/Ang-(1-7)/MasR Axis.

Electroacupuncture (EA) can effectively alleviate anxiety disorders and memory impairments caused by various neurodegenerative diseases; however, the molecular mechanisms underlying its neuroprotective effects are unclear. Previous studies have shown that the renin-angiotensin system (RAS) comprises of two axes with mutual antagonism: the classical angiotensin converting enzyme/angiotensin II/angiotensin II type 1 receptor (ACE/Ang II/AT1R) axis and the protective angiotensin converting enzyme 2/angiotensin-(1-7)/Mas receptor (ACE2/Ang-(1-7)/MasR) axis. In this study, we observed that chronic cerebral hypoperfusion (CCH) mediated anxiety-like behavior and memory impairments in spontaneously hypertensive rats (SHR) via upregulation of the hippocampal classical axis (ACE/Ang II/AT1R) and the partial hippocampal protective axis (ACE2/Ang-(1-7)). However, Ang II levels were much higher than those of Ang-(1-7), indicating that the ACE/Ang II/AT1R axis plays a dominant role in the comorbidity of CCH and hypertension. Moreover, candesartan cilexetil (Canc) and perindopril (Peril) were used as positive control drugs. We found that EA, Canc, and Peril attenuated CCH-induced anxiety-like behavior and memory impairments in SHR, potentially via downregulation of the hippocampal classical axis (ACE/Ang II/AT1R) and upregulation of the whole hippocampal protective axis (ACE2/Ang-(1-7)/MasR). These results suggest that EA therapy for CCH with hypertension may be mediated by two hippocampal RAS axes.

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.

Effects of Electroacupuncture on Pain Memory-Related Behaviors and Synchronous Neural Oscillations in the Rostral Anterior Cingulate Cortex in Freely Moving Rats.

Our previous studies have confirmed that electroacupuncture (EA) can effectively intervene in pain memory, but the neural mechanism involved remains unclear. In this study, we observed the effects of EA in regulating pain memory-related behaviors and synchronous neural oscillations in the rostral anterior cingulate cortex (rACC). During nociceptive behavioral testing, pain memory induced a nonpain stimulus that spurred a neural oscillatory reaction similar to that caused by pain stimuli in the rACC. After EA, nonpain stimuli did not induce decreased neural oscillatory activity in the rACC until the presentation of pain stimuli. During aversive behavioral testing, EA, through the downregulation of theta power, inhibited the retrieval of aversive memory and relieved pain memory-induced aversive behaviors. These changes of oscillatory activity may be the hallmarks of EA therapy for pain memory.

Proteomics analysis of the amygdala in rats with CFA-induced pain aversion with electro-acupuncture stimulation.

BACKGROUND: Clinical patients suffering from pain usually exhibit aversion to pain-associated environments (pain aversion). Electro-acupuncture (EA) has been proven to be effective for the treatment of pain aversion in our previous studies. The amygdala could have substantial consequences on emotion and pain consolidation as well as general pain aversion behavior, however, the underlying mechanism remains unclear. PURPOSE: The current study was performed to investigate Isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative proteomic analysis of the amygdala in rats with complete Freund's adjuvant (CFA)-induced pain aversion, and comprehensive analysis of protein expression were performed to explore the underlying mechanism by which EA affects pain aversion. MATERIALS AND METHODS: Inflammatory pain was induced with an intraplantar injection of 100 µL of CFA in the plantar surface of the left hind paw of the male Spragure-Dawley (SD) rats. Then the CFA-induced conditioned place aversion (C-CPA) test was performed. EA stimulation on the bilateral Zusanli and Sanyinjiao acu-points was used for 14 days and the EA stimulation frequency is 2 Hz. Based on iTRAQ-based proteomics analysis, we investigated the protein expression in the amygdala. RESULTS: EA can increase the paw withdrawal threshold in inflammatory pain induced by noxious stimulation. A total of 6319 proteins were quantified in amygdala. Of these identified proteins, 123 were identified in the pain aversion group relative to those in the saline group, and 125 significantly altered proteins were identified in the pain aversion + EA group relative to the pain aversion group. A total of 11 proteins were found to be differentially expressed in the amygdala of pain aversion and EA-treated rats. The expression of three proteins, glyceraldehyde-3-phosphate dehydrogenase, glutamate transporter-1, and p21-activated kinase 6, were confirmed to be consistent with the results of the proteome. CONCLUSION: Our investigation demonstrated the possible mechanism of central nerve system by which EA intervetion on pain aversion.

[Feasibility of Joint Application of Techniques of Optogenetics and Neuroelectrophysiology to Research of Acupuncture Analgesia].

Since the invention of optogenetic technology, it has greatly promoted the development of neuroscience. Currently, optogenetic approaches have been mostly used to map neural circuits and new neuropharmacology but are rarely seen in the research field of acupuncture analgesia. The mechanism of neural circuits contributing to acupuncture analgesia, an important research hotspot in recent years, has not been fully determined. The optogenetic techniques can be used to modulate and control specific cells, provides highly precise spatial and temporal resolution, is repeatable, and may functionally dissect neuronal networks in vivo. The neuronal activities and their information transmission, processing and storage in intercluster neural networks in different brain regions, and the correlation between behavioral changes and electrical activities of neurons in vivo studies are mainly captured by the implanted microelectropode array, etc. If these two (or more) approaches are combined together, it is definitely and highly helpful to reveal the driving dynamics of neural circuits, plasticity and temporal-spatial activity mode of neurons, as well as behavioral reactions of animals with chronic pain during acupuncture analgesia and may open a new prospect for the application of acupuncture analgesia study.

5-HT in the dorsal raphe nucleus is involved in the effects of 100-Hz electro-acupuncture on the pain-depression dyad in rats.

The pain-depression dyad is becoming widespread in the clinic and is attracting increasing attention. A previous study by our group found that 100-Hz electro-acupuncture (EA), but not 2-, 50- and 2/100-Hz EA, was effective against the reserpine-induced pain-depression dyad. This finding is in contrast to the fact that low-frequency EA is commonly used to treat supraspinal-originating diseases. The present study aimed to investigate the mechanism underlying the effects of 100-Hz EA on the pain-depression dyad. Repeated reserpine injection was found to induce allodynia and depressive behaviors in rats. It decreased 5-hydroxytryptamine (5-HT) levels and immunoreactive expressions in the dorsal raphe nucleus (DRN). 100-Hz EA alleviated the pain-depression dyad and upregulated 5-HT in the DRN of reserpine-injected rats. Intracerebroventricular injection of para-chlorophenylalanine, an inhibitor of 5-HT resynthesis, suppressed the upregulation of 5-HT in the DRN by 100-Hz EA and partially counteracted the analgesic and anti-depressive effects of 100-Hz EA. The present study was the first to demonstrate that 5-HT in the DRN is involved in mediating the analgesic and anti-depressive effects of 100-Hz EA on the pain-depression dyad. This finding provided a scientific basis for high-frequency EA as a potential treatment for the pain-depression dyad.

Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model.

Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

Electroacupuncture alleviates retrieval of pain memory and its effect on phosphorylation of cAMP response element-binding protein in anterior cingulate cortex in rats.

BACKGROUND: Recent evidence suggests that persistent pain and recurrent pain are due to the pain memory which is related to the phosphorylation of cAMP response element-binding protein (p-CREB) in anterior cingulate cortex (ACC). Eletroacupuncture (EA), as a complementary Chinese medical procedure, has a significant impact on the treatment of pain and is now considered as a mind-body therapy. METHODS: The rat model of pain memory was induced by two injections of carrageenan into the paws, which was administered separately by a 14-day interval, and treated with EA therapy. The paw withdrawal thresholds (PWTs) of animals were measured and p-CREB expressions in ACC were detected by using immunofluorescence (IF) and electrophoretic mobility shift assay (EMSA). Statistical comparisons among different groups were made by one-way, repeated-measures analysis of variance (ANOVA). RESULTS: The second injection of carrageenan caused the decrease of PWTs in the non-injected hind paw. EA stimulation applied prior to the second injection, increased the values of PWTs. In ACC, the numbers of p-CREB positive cells were significantly increased in pain memory model rats, which were significantly reduced by EA. EMSA results showed EA also down-regulated the combining capacity of p-CREB with its DNA. Furthermore, the co-expression of p-CREB with GFAP, OX-42, or NeuN in ACC was strengthened in the pain memory model rats. EA inhibited the co-expression of p-CREB with GFAP or OX-42, but not NeuN in ACC. CONCLUSIONS: The present results suggest the retrieval of pain memory could be alleviated by the pre-treatment of EA, which is at least partially attributed to the down-regulated expression and combining capacity of p-CREB and the decreased expression of p-CREB in astrocytes and microglia cells.

Strong Manual Acupuncture Stimulation of "Huantiao" (GB 30) Reduces Pain-Induced Anxiety and p-ERK in the Anterior Cingulate Cortex in a Rat Model of Neuropathic Pain.

Persistent neuropathic pain is associated with anxiety. The phosphorylation of extracellular signal-regulated kinase (p-ERK) in the anterior cingulate cortex (ACC) plays an important role in pain-induced anxiety. Acupuncture is widely used for pain and anxiety. However, little is known about which acupuncture technique is optimal on pain-induced anxiety and the relationship between acupuncture effect and p-ERK. The rat model was induced by L5 spinal nerve ligation (SNL). Male adult SD rats were randomly divided into control, SNL, strong manual acupuncture (sMA), mild manual acupuncture (mMA), and electroacupuncture (EA) group. Bilateral "Huantiao" (GB 30) were stimulated by sMA, mMA, and EA, respectively. The pain withdrawal thresholds (PWTs) and anxiety behavior were measured, and p-ERK protein expression and immunoreactivity cells in ACC were detected. PWTs increased significantly in both sMA and EA groups. Meanwhile, anxiety-like behavior was improved significantly in the sMA and mMA groups. Furthermore, the overexpression of p-ERK induced by SNL was downregulated by strong and mild manual acupuncture. Therefore, strong manual acupuncture on bilateral "Huantiao" (GB 30) could be a proper therapy relieving both pain and pain-induced anxiety. The effect of different acupuncture techniques on pain-induced anxiety may arise from the regulation of p-ERK in ACC.

[Effect of mild and strong manual acupuncture stimulation of "Huantiao" (GB 30) on mechanical pain thresholds and extracellular signal-regulated kinase protein expression in spinal dorsal horns in rats with neuropathic mirror-image pain].

OBJECTIVE: To observe the effect of different intensities of manual acupuncture (MA) stimulation on mechanical pain thresholds (PTs) and the expression of phosphorylated extracellular signal-regulated kinases (p-ERK) in lumbar spinal dorsal horn regions in rats with neuropathic mirror-image pain, so as to explore its mechanisms underlying analgesia. METHODS: Forty male SD rats were equally and randomly divided into control, spinal nerve ligation (SNL) model, mild MA-stimulation, and strong MA-stimulation groups. Neuropathological pain model was established by ligature of the spinal nerve (L 5). Three days after the SNL, bilateral "Huantiao" (GB 30) were stimulated by rotating the thin (0.22 mm x 13 mm) or thick (0.3 mm x 13 mm) filiform needles at a frequencies of 60 times/min or 180 times/min and at an angle of 180 degrees or 360 degrees for 2 min for rats in the mild and strong MA-stimulation groups, respectively, followed by remaining the needle in place for 30 min. The mechanical PTs were measured before and after SNL. The expression of p-ERK protein in bilateral dorsal horn regions of the lumbar spinal cord (L4- L 6) was detected by Western blot. RESULTS: In comparison with the control group, the mechanical PTs were significantly decreased beginning from the 3rd day on after SNL on the affected side and from the 7th day on after SNL on the healthy hindpaw (P < 0.05), simultaneously, p-ERK protein expression levels of dorsal horn regions on both sides of the spinal cord were considerably up-regulated on the 12th day (P < 0.05). Compared with the model group, the PTs of the affected hindpaw and the healthy hindpaw were significantly increased on the 7th and 12th day in the strong MA-stimulation group (P < 0.05, P < 0.01), whereas pERK expression levels in the bilateral spinal dorsal horn regions were obviously down-regulated in the strong MA-stimulation group (P < 0.05). No significant differences were found between the model and mild MA-stimulation groups in the PTs of bilateral hindpaws and p-ERK expression levels of the bilateral spinal dorsal horn regions (P > 0.05) except the PTs of the healthy hindpaw on 7th day (P < 0.05). CONCLUSION: Strong MA-stimulation can alleviate neuropathic mirror-image pain in SNL rats, which is closely related to its effect in down-regulating the expression of p-ERK in the bilateral spinal dorsal horn regions.