Electroacupuncture may alleviate diabetic neuropathic pain by inhibiting the microglia P2X4R and neuroinflammation.

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1ß and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability.

Electroacupuncture Alleviates Diabetic Neuropathic Pain and Downregulates p-PKC and TRPV1 in Dorsal Root Ganglions and Spinal Cord Dorsal Horn.

Diabetic neuropathic pain (DNP) is a common complication of diabetes. Streptozotocin (STZ)-induced changes of protein in dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) are critical for DNP genesis. However, which proteins change remains elusive. Here, the DNP model was established by a single intraperitoneal injection of STZ, accompanied by increased fasting blood glucose (FBG), decreased body weight (BW), and decreased paw withdrawal latency (PWL). Proteins change in L4-L6 DRGs and SCDH of rats were detected. Western blot and immunofluorescence results showed that expression levels of phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1), Substance P (SP) and calcitonin gene-related peptide (CGRP) in the DRG and the SCDH of rats were increased after STZ injection. A preliminary study from our previous study showed that 2 Hz electroacupuncture (EA) effectively alleviates DNP. However, the analgesic mechanism of EA needs further elucidation. Here, EA at the bilateral Zusanli (ST36) and KunLun (BL60) acupoints was applied for one week, and to investigate the effect on DNP. EA reversed thermal hyperalgesia in DNP rats and downregulated the expression of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH.

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7.

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.

Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 µL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,ß-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,ß-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,ß-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.

Amygdalar κ-opioid receptor-dependent upregulating glutamate transporter 1 mediates depressive-like behaviors of opioid abstinence.

Opiates produce a strong rewarding effect, but abstinence from opiate use emerges with severe negative emotions. Depression is one of the most frequent emotion disorders associated with opiate abstinence, which is thought to be a main cause for relapse. However, neurobiological bases of such an aversive emotion processing are poorly understood. Here, we find that morphine abstinence activates κ-opioid receptors (KORs) by increasing endogenous KOR ligand dynorphin expression in the amygdala, which in turn facilitates glutamate transporter 1 (GLT1) expression by activation of p38 mitogen-activated protein kinase (MAPK). Upregulation of GLT1 expression contributes to opiate-abstinence-elicited depressive-like behaviors through modulating amygdalar glutamatergic inputs to the nucleus accumbens (NAc). Intra-amygdala injection of GLT1 inhibitor DHK or knockdown of GLT1 expression in the amygdala significantly suppresses morphine-abstinence-induced depressive-like behaviors. Pharmacological and pharmacogenetic activation of amygdala-NAc projections prevents morphine-abstinence-induced behaviors. Overall, our study provides key molecular and circuit insights into the mechanisms of depression associated with opiate abstinence.

Anxiolytic effect of GABAergic neurons in the anterior cingulate cortex in a rat model of chronic inflammatory pain.

Chronic pain easily leads to concomitant mood disorders, and the excitability of anterior cingulate cortex (ACC) pyramidal neurons (PNs) is involved in chronic pain-related anxiety. However, the mechanism by which PNs regulate pain-related anxiety is still unknown. The GABAergic system plays an important role in modulating neuronal activity. In this paper, we aimed to study how the GABAergic system participates in regulating the excitability of ACC PNs, consequently affecting chronic inflammatory pain-related anxiety. A rat model of CFA-induced chronic inflammatory pain displayed anxiety-like behaviors, increased the excitability of ACC PNs, and reduced inhibitory presynaptic transmission; however, the number of GAD65/67 was not altered. Interestingly, intra-ACC injection of the GABAAR agonist muscimol relieved anxiety-like behaviors but had no effect on chronic inflammatory pain. Intra-ACC injection of the GABAAR antagonist picrotoxin induced anxiety-like behaviors but had no effect on pain in normal rats. Notably, chemogenetic activation of GABAergic neurons in the ACC alleviated chronic inflammatory pain and pain-induced anxiety-like behaviors, enhanced inhibitory presynaptic transmission, and reduced the excitability of ACC PNs. Chemogenetic inhibition of GABAergic neurons in the ACC led to pain-induced anxiety-like behaviors, reduced inhibitory presynaptic transmission, and enhanced the excitability of ACC PNs but had no effect on pain in normal rats. We demonstrate that the GABAergic system mediates a reduction in inhibitory presynaptic transmission in the ACC, which leads to enhanced excitability of pyramidal neurons in the ACC and is associated with chronic inflammatory pain-related anxiety.

[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.

[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 combined with PNF on proprioception and motor function of lower limbs in stroke patients: a randomized controlled trial].

OBJECTIVE: To observe the clinical efficacy and correlation of electroacupuncture combined with proprioceptive neuromuscular facilitation (PNF) on proprioception and motor function of lower limbs in stroke patients. METHODS: A total of 96 stroke patients were randomized into an electroacupuncture (EA) group, a PNF group and a combination group, 32 cases in each one. In the EA group, acupuncture was applied at cephalic motor and sensory areas, Huantiao (GB 30), Yanglingquan (GB 34), Xuanzhong (GB 39), Zusanli (ST 36) and Sanyinjiao (SP 6) on affected side, and electroacupuncture was adopted at Yanglingquan (GB 34) and Xuanzhong (GB 39), continuous wave and 2 Hz in frequency for 20 min, once every day. In the PNF group, PNF was performed for 20 min, once a day. In the combination group, electroacupuncture was given before PNF, once a day. 4 weeks as one course and totally 3 courses were required, the effect was followed up after half a year. Before treatment, after 4, 8, 12 weeks of treatment and in follow-up, the average trace error (ATE) and Time, the scores of Fugl-Meyer scale (FMA) and modified Barthel index (MBI) were observed in the 3 groups. Correlation analysis between ATE, Time and FMA was performed. RESULTS: ① The total effective rate in the combination group was 90.3% (28/31), which was superior to 64.5% (20/31) in the EA group and 62.5% (20/32) in the PNF group (P<0.05). ②After 4, 8, 12 weeks of treatment and in follow-up, the ATE and Time in the 3 groups were reduced compared with before treatment (P<0.05, P<0.01). After 8, 12 weeks of treatment and in follow-up, the ATE in the combination group were lower than the EA group and the PNF group (P<0.05, P<0.01). After 12 weeks of treatment and in follow-up, the Time in the combination group were lower than the EA group and the PNF group (P<0.05, P<0.01). ③After 4, 8, 12 weeks of treatment and in follow-up, the FMA scores in the EA group and the combination group were increased compared with before treatment (P<0.01). After 8, 12 weeks of treatment and in follow-up, the FMA scores in the PNF group were increased compared with before treatment (P<0.01). After 8, 12 weeks of treatment and in follow-up, the FMA scores in the combination group were higher than the EA group and the PNF group (P<0.05, P<0.01). ④After 4, 8, 12 weeks of treatment and in follow-up, the MBI scores in the 3 groups were increased compared with before treatment (P<0.01). After 8, 12 weeks of treatment and in follow-up, the MBI scores in the combination group were higher than the EA group (P<0.01). After 12 weeks of treatment and in follow-up, the MBI scores in the combination group were higher than the PNF group (P<0.01). ⑤ The correlation coefficients of ATE, Time and FMA were from 0.4 to 0.75 (P<0.05), suggesting a moderate intensity correlation. CONCLUSION: Electroacupuncture, PNF and combination therapy can improve proprioception and motor function of lower limbs and activities of daily living, and combination therapy has a better effect. Proprioception and motor function have a strong correlation in the recovery of stroke patients.

[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.

Suppressing PKC-dependent membrane P2X3 receptor upregulation in dorsal root ganglia mediated electroacupuncture analgesia in rat painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is a common and troublesome diabetes complication. Protein kinase C (PKC)-mediated dorsal root ganglia (DRG) P2X3 receptor upregulation is one important mechanism underlying PDN. Accumulating evidence demonstrated that electroacupuncture (EA) at low frequency could effectively attenuate neuropathic pain. Our previous study showed that 2-Hz EA could relieve pain well in PDN. The study aimed to investigate whether 2-Hz EA relieves pain in PDN through suppressing PKC-mediated DRG P2X3 receptor upregulation. A 7-week feeding of high-fat and high-sugar diet plus a single injection of streptozotocin (STZ) in a dose of 35 mg/kg after a 5-week feeding of the diet successfully induced type 2 PDN in rats as revealed by the elevated body weight, fasting blood glucose, fasting insulin and insulin resistance, and the reduced paw withdrawal threshold (PWT), as well as the destructive ultrastructural change of sciatic nerve. DRG plasma membrane P2X3 receptor level and DRG PKC expression were elevated. Two-hertz EA failed to improve peripheral neuropathy; however, it reduced PWT, DRG plasma membrane P2X3 receptor level, and DRG PKC expression in PDN rats. Intraperitoneal administration of P2X3 receptor agonist αß-meATP or PKC activator phorbol 12-myristate 13-acetate (PMA) blocked 2-Hz EA analgesia. Furthermore, PMA administration increased DRG plasma membrane P2X3 receptor level in PDN rats subject to 2-Hz EA treatment. These findings together indicated that the analgesic effect of EA in PDN is mediated by suppressing PKC-dependent membrane P2X3 upregulation in DRG. EA at low frequency is a valuable approach for PDN control.

[Correlation Between Referred Pain Region and Sensitized Acupoints in Patients with Stable Angina Pectoris and Distribution of Sensitized Spots in Rats with Myocardial Ischemia].

OBJECTIVE: To observe the correlation between the referred pain regions of stable angina pectoris (SAP) and the acupoints in coronary heart disease (CHD) patients and to investigate the rule of regional sensitized point distribution in rats. METHODS: A total of 1 046 CHD patients with SAP from 8 hospitals in China were recruited in the present study. The tenderness was palpated along the left and right chest, back, shoulder, upper limb, etc. by a specially-assigned researcher in each hospital. Among them, 77 patients accepted pain threshold (PT) measurement by using a hand-held esthesiometer. In animal experiments, 14 SD rats were subjected to occlusion of the left anterior descending branch of the left coronary artery for 4 h for establishing myocardial ischemia (MI) model, and other 4 normal rats were used as the sham-operation control group. Four hours after MI, all the rats accepted tail venous injection of 5% Evans blue (50 mg/kg) for examining the distribution of the blue dye exudation spots at the body surface where the mechanical PT was also detected by a von Frey. RESULTS: In 1 046 CHD patients, 987 (94.36%) were found to have at least one tenderness spot. The tenderness spots were found at the left chest (87.47%), right chest (13.67%), left arm (ulnar side, 41.30%), right upper limb (4.68%), left shoulder back (30.21%), right shoulder back (7.07%), etc., accompanied with rash or pigmentation, subcutaneous induration, cord-like tissue contracture, skin sag, etc. The mechanical PT level was significantly lower at the tenderness spots of the left upper limb than at non-tender points of the right upper limb in CHD patients (P<0.001). Tenderness and cutaneous abnormal changes in angor pectoris patients distributed mostly on the left chest, back, shoulder and upper limb, and some also on the right. Tender points scattered on, near or outside acupoints. A similar distribution of the blue exudation spots and lower mechanical PT spots were found in MI rats, but not in sham-MI rats. CONCLUSION: In the case of MI, a regular "referred sensitization" response frequently occurs in the dermatomere area innervated by the corresponding segments (T 1－T 5) in both CHD patients and MI rats, which may be closely associated with the formation of acupoints in ancient China.

Alleviating Mechanical Allodynia and Modulating Cellular Immunity Contribute to Electroacupuncture's Dual Effect on Bone Cancer Pain.

HYPOTHESIS: Electroacupuncture (EA) has been used as an alternative analgesic therapy for hundreds of years, yet its analgesic potency and therapeutic advantage against bone cancer pain (BCP) in comparison with morphine remains unclear. This study aimed to investigate the effects of EA on mechanical allodynia and cellular immunity of BCP rats, and to further explore the potential mechanism. METHODS: The BCP model was established by implanting Walker 256 mammary gland carcinoma cells into the left tibia of adult female Sprague-Dawley rats. EA (dilatational wave, 2/100 Hz, 0.5 mA-1mA-1.5 mA for 10 minutes each intensity) was applied bilaterally to Zusanli (ST 36) and Kunlun (BL 60) for 30 minutes. Both EA stimulation and morphine (10 mg/kg, intraperitoneally) was given once every other day. Naloxone (0.3 mg/kg, intraperitoneally) was injected at 30 minutes prior to EA. Mechanical allodynia were demonstrated by paw withdrawal thresholds (PWTs) which measured by dynamic plantar aesthesiometer. T cell proliferation, percentage of CD3+, CD4+ and CD8+ T lymphocytes in spleen as well as expression of interleukin-2 (IL-2) in plasma were detected by WST-8, flow cytometry, and enzyme-linked immunosorbent assay technique, respectively. RESULTS: An intratibial inoculation of Walker 256 mammary gland carcinoma cells significantly decreased PWTs to mechanical stimuli. EA stimulation alleviated mechanical allodynia in BCP rats, and the analgesic potency of EA was weaker than that of morphine. In contrast to morphine, EA stimulation of BCP rats increased splenic concanavalin A (Con A)-induced T cell proliferation and plasma IL-2 content, as well as increased the percentages of splenic CD3+CD4+ and CD3+CD8+ T cell subsets. Moreover, both the analgesic effect and the partial immunomodulation of EA were suppressed by an intraperitoneal injection of naloxone. CONCLUSION: EA could significantly alleviate BCP-induced mechanical allodynia. Although the analgesic effect of EA was weaker than that of morphine, EA had an immunomodulation effect on cellular immunity. Both analgesic and immunomodulatory effect of EA might share the same mechanism via the opioid-mediated pathway, which needs further investigation.

[New Trains of Thoughts About Acupuncture Analgesia-Acupuncture Analgesia Feb Involve Multi-dimensional Regulation of Pain].

With the development of pain study, researchers gradually recognized that pain is composed of three main dimensions, namely "sensory-discriminative" "affective-motivational" and "cognitive-evaluative" which influence each other and are also independent from each other. Pain study has shifted away from focusing on the single mode of nociception to the multi-dimensional mode of sensory-affection-cognition. It is held early in traditional Chinese medicine that "when there is a stoppage, there is a pain" and a worsening disease Feb induce depression, which has already shown a multi-dimensional recognition about pain. Acupuncture therapy has been considered to be an effective adjuvant approach for relieving pain. In the present paper, the authors introduced applicability of acupuncture analgesia by modulating the abovementioned multi-dimensions of pain from the following 4 aspects:1) multi-dimensions of pain and related brain regions; 2) recognitions of traditional Chinese medicine about pain; 3) development of researches on acupuncture analgesia, including a) balancing activities of endogenous analgesic and algogenic substances, and triggering intracellular mitogen-activated protein kinase (MAPK) signaling to reduce algesia, b) improving psychological symptoms of patients with depression, anxiety, insomnia, etc., c) modulating functional activities of some common brain regions (as hippocampus, anterior cingutate, frontal lobe of cerebral cortex, etc.) sharing both pain information and learning-memory processing. Hence, the authors hold that if the clinical study and application and experimental researches conducted on the underlying mechanisms of acupuncture analgesia extend towards the multi-dimensions of pain, a series of new concepts or thoughts will be brought out, thereby possibly opening a bright applicable prospect for acupuncture analgesia.

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.

Effect of Electroacupuncture on the NTS is modulated primarily by acupuncture point selection and stimulation frequency in normal rats.

BACKGROUND: The effect of electroacupuncture (EA) is affected by both the acupuncture point selection and the frequency of stimulation. However, little is known regarding acupuncture point and simulation frequency selection. Neuronal activation of the nucleus of the solitary tract (NTS) is one of the important targets of EA for modulating gastrointestinal function. This study investigated the effects of various combinations of EA frequencies and acupuncture points on NTS neurons. METHODS: Rats were randomly divided into normal, 2 Hz EA, 100 Hz EA and the alternate 2/100 Hz EA groups. Then rats in each group were randomly divided into the following two subgroups according to the acupuncture point: ST 36 group and ST 25 group. All the rats underwent electrode implantation surgery. Rats in all EA groups received one treatment with EA (a constant square wave at, 2 Hz,100 Hz or 2/100 Hz frequencies with intensities ranging from 1 to 2 mA), and NTS neuronal activation was recorded before and after EA treatment. Finally, to confirm the effect of EA on the NTS, minimal acupuncture was administered and its effect on NTS was detected. RESULTS: ST 36 stimulated with 2 Hz EA significantly increased the population of excited NTS neurons and spike frequency. However, ST 36 stimulated with 100 Hz or 2/100 Hz EA produced only a transient effect on the activity of NTS neurons and did not induce any effect on the spike frequency. Furthermore, the excitatory effect of 100 Hz or 2/100 Hz EA on NTS neurons in the ST 36 group was lower than 2 Hz EA at the same point. When applied to ST 25, 2 Hz EA had no significant excitatory effect on NTS neurons or spike frequency. However, 100 Hz EA or 2/100 Hz EA at ST 25 decreased both NTS neuronal excitability and spike frequency. By comparing the effects of different EA combinations, it was shown 2 Hz EA applied to ST 36 had the strongest excitatory effect on NTS neurons, while 100 Hz EA applied to ST 25 had the greatest inhibitory effect. Minimal acupuncture stimulation produced no effect on NTS neurons. CONCLUSION: EA's effects on NTS were mainly affected by the acupuncture point selection, but the frequency of EA also played a role. Different combinations of acupuncture points and frequency selection may lead to different EA effects on NTS neuronal excitability.

Electroacupuncture treatment partly promotes the recovery time of postoperative ileus by activating the vagus nerve but not regulating local inflammation.

Postoperative ileus (POI) after abdominal surgery significantly lowers the life quality of patients and increase hospital costs. However, few treatment strategies have successfully shortened the duration of POI. Electroacupuncture (EA) is a modern way of administering acupuncture and widely used in various gastrointestinal (GI) diseases in the world. Here, we studied the effect of EA on POI and its underlying mechanisms. Intestinal manipulation resulted in significant delays of GI transit, colonic transit and gastric emptying. Surgery also up-regulated c-fos in nucleus of the solitary tract (NTS) and induced inflammation response in the small intestine. Further, operation and inhale anesthesia inhibited NTS neuron excitation duration for the whole observation time. EA administered at ST36 indeed shortened the recovery time of GI and colonic transit, and significantly increased the gastric emptying. EA also significantly activated the NTS neurons after operation. However, there was no anti-inflammation effect of EA during the whole experiment. Finally, atropine blocked the regulatory effect of EA on GI function, when it was injected after surgery, but not before surgery. Thus, the regulatory effect of EA on POI was mainly mediated by exciting NTS neurons to improve the GI tract transit function but not by activating cholinergic anti-inflammatory pathway.

[Suggestions for standardized management of nomenclature and classification of neonatal diseases].

Nomenclature and classification of diseases are not only related to clinical diagnosis and treatment, but also involved in the fields such as management and exchange of medical information, medical expense payments, and medical insurance payment. In order to standardize clinical physicians' diagnostic and treatment activities, medical records, and the first page of medical records, this article elaborates on the basic principles and methods for nomenclature and classification of diseases with reference to international nomenclature of diseases and international classification of diseases. Meanwhile, in view of the problems in clinical practice, this article proposes the classification of neonatal diseases, the basic procedure and writing rules in the diagnosis of neonatal diseases, and death diagnosis principles.

Effect of systemic injection of heterogenous and homogenous opioids on peripheral cellular immune response in rats with bone cancer pain: A comparative study.

Exogenous and endogenous opioids have been shown to modulate the immune system. Morphine-induced immunosuppression has been investigated extensively. However, the immune-regulating function of endogenous opioid peptides is unclear. The present study aimed to evaluate the difference in effects on cellular immune function between recombinant rat ß-endorphin (ß-EP; 50 µg/kg) and plant source morphine (10 mg/kg) via intraperitoneal injection treatment in a rat model of bone cancer pain. Walker 256 cells were injected into a tibial cavity injection to establish the bone cancer pain model. The paw withdrawal thresholds and body weights were measured prior to surgery, at 6 days after surgery, and following 1, 3,6 and 8 treatments. The spleen cells were harvested for detection of T cell proliferation, natural killer (NK) cell cytotoxicity, and the relative quantities of T cell subtypes (CD3+, CD4+ and CD8+ cells). Plasma levels of interleukin-2 (IL-2) were also determined. It was found that single or multiple treatments with ß-EP (a homogenous opioid peptide) and morphine (a heterogenous opioid) had good analgesic effects on bone cancer pain, while the analgesia provided by morphine was stronger than that of ß-EP. Treatment with ß-EP 3, 6 and 8 times increased the body weight gain in the rat model of bone cancer pain, while morphine treatment had on effect on it. With regard to immunomodulatory functions, ß-EP treatment increased T cell proliferation and NK cell cytotoxicity, and increased the relative quantities of T cell subtypes, but no effect on T cell secretion. However, morphine treatment decreased T cell proliferation and the levels of T cell subtypes. These data indicate that opioids from different sources have different effects on cellular immune function in vivo. A small dose of homogenous opioid peptide exhibited positive effects (analgesia and immune enhancement) on cancer pain. These results provide experimental evidence supporting the exploitation of human opioids for the treatment of cancer pain.