[Electroacupuncture and transcutaneous electrical acupoint stimulation relieve muscular inflammatory pain by activating afferent nerve fibers in different layers of "Liangqiu"(ST34) in rats].

OBJECTIVE: To compare the effect of electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS) of skin and muscle layers of "Liangqiu" (ST34) area on inflammatory muscular pain in rats, so as to study the role of different-layer afferent nerve fibers in acupuncture analgesia. METHODS: A total of 120 male SD rats were used in the present study, including 8 rats used for determining the excitatory threshold of Aδ(Ta) and C (Tc) afferent nerve fibers, 48 employed for comparing the analgesic effect of EA and TEAS at intensities of Ta and Tc, and 64 for observing the effect of EA and TAES stimulation of ipsilateral (Ipsi), contralateral (Contra) ST34 and ipsilateral LI4 at Ta and Tc intensities. Inflammatory muscle pain was induced by injection of complete Freund's adjuvant (CFA) into the right biceps femoris muscle. In the second part of the present study, 48 rats were randomly and equally divided into control, model, TEAS-Ta, TEAS-Tc, EA-Ta and EA-Tc groups, while in the 3rd part, 64 rats were randomly and equally divided into control, model, Ipsi-ST34-TEAS, Contra-ST34-TEAS, Ipsi-ST34-EA, Contra-ST34-EA, Ipsi-LI4-TEAS and Ipsi-LI4-EA groups. TEAS or EA was applied to the skin and muscle layers, respectively. Before and after modeling, the animal was forced to stand on a bipedal equilibrator, the difference in body mass distribution of both feet (bearing difference) was used to assess the pain severity. The frequency of myoelectrical discharges of the right bicep femoris muscle in responding to electrical stimulation of the spot between the 4th and 5th toes of the ipsilateral hindlimb at an intensity of two-folds of C-fiber excitatory threshold was recorded. RESULTS: 1) The bearing difference between the bilateral hindlimbs was markedly higher in the model group than in the control group (P<0.01), and significantly lower at the 2nd and 3rd day in the EA-Ta and EA-Tc, and TEAS-Tc groups relevant to the model group (P<0.05, P<0.01). 2) The frequency of C-fiber reflex induced electromyogram (EMG) activities were significantly decreased at 0 and 1 min after TEAS of both ipsilateral ST34 at Tc, and 0 min after TEAS of the contralateral ST34 at Tc (rather than at Ta and not LI4 even at Tc) in comparison with pre-TEAS, and 0, 1 and 2 min after TEAS of ipsilateral ST34 at Tc, and 0 min after TEAS of contra-ST34 at Tc compared with the model group, respectively (P<0.01, P<0.05). In comparison with pre-EA, the frequency of C-fiber reflex induced EMG activities were significantly decreased at 0 and 1 min after EA of the ipsilateral ST34 at Ta, and 0 min after EA of the contra-ST34 at Ta. In addition, the frequency of C-fiber reflex induced EMG activities were decreased at 0, 1 and 2 min after EA of the ipsilateral ST34 at Tc, and 0 and 1 min after EA of the contra-ST34 at Tc, as well as 0 min after EA of LI4 at Tc (P<0.01, P<0.05).In comparison with the model group, the frequency of C-fiber reflex induced EMG activities are significantly decreased at 0, and 1 min after EA of the ipsilateral ST34 at Ta, and 0 min after EA of the contra-ST34 at Ta. In addition, the frequency of C-fiber reflex induced EMG activities were decreased at 0, 1 ,2,and 3 min after EA of the ipsilateral ST34 at Tc, and 0 and 1 min after EA of the contra-ST34 at Tc, as well as 0 min after EA of LI4 at Tc, respectively (P<0.01, P<0.05). CONCLUSION: TEAS-ST34 at Tc and EA-ST34 at both Ta and Tc can alleviate pain behavior in inflammatory pain rats, which may be related to its effect in activating the afferent nerve fiber in different layers of ST34 area.

[Electroacupuncture and transcutaneous electrical acupoint stimulation with different intensities relieve muscular of inflammatory pain of the rats].

OBJECTIVE: To observe and compare the ameliorate effects of electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS) with different intensities on inflammatory muscle pain, so as to confirm the role of different afferent nerve fibers in different layers (skin or muscle) in pain relief of acupuncture. METHODS: The intensities of the thresholds of A (Ta) and C (Tc) were selected for TEAS and EA. TEAS and EA were applied on the layer of skin and muscle of "Liangqiu" (ST34), respectively. Forty-eight rats were randomly divided into control, model, TEAS-Ta, TEAS-Tc, EA-Ta and EA-Tc groups (n=8 in each group). The inflammatory muscle pain model was established by complete Freund's adjuvant (CFA) injection into the right biceps femoris muscle. EA or TEAS was applied once a day for 3 days. The weight difference between the hind limbs and the abnormal electromyography (EMG) activities were observed as an index for pain of the rats. RESULTS: After modeling, the weight difference between the hind limbs was increased markedly in the model group relevant to the control group (P<0.01). Compared with the model group, the weight difference between the hind limbs in the TEAS-Tc, EA-Ta, and EA-Tc groups was significantly decreased (P<0.05, P<0.01). Compared with that before intervention, the area under the curve and discharge frequency of abnormal EMG of rats in the TEAS-Tc and EA-Ta groups were significantly reduced after intervention (P<0.01, P<0.05), while those in the EA-Tc group were significantly increased (P<0.05). After the intervention, compared with the TEAS-Ta group, the inhibition rate of the area under the curve and the discharge frequency of the abnormal EMG in the TEAS-Tc group increased significantly (P＜0.05); and those were also increased in the EA-Ta group when compared with the EA-Tc group (P<0.01). CONCLUSION: TEAS with Tc or EA with Ta in the muscle layer can alleviate the pain and inhibit the abnormal EMG in inflammatory muscle pain rats. It is indicated that local pain relief by acupuncture was related to the afferent nerve fiber in different layer with different intensities.

GABAergic Inhibition of Spinal Cord Dorsal Horns Contributes to Analgesic Effect of Electroacupuncture in Incisional Neck Pain Rats.

BACKGROUND: Acupuncture has shown to be effective in relieving post-surgical pain. Nonetheless, its underlying mechanisms remain largely unknown. In the present study, we investigated the effect of electroacupuncture (EA) on the expression of GABA, GABA-A receptor (R) and GABA-BR in the spinal cord dorsal horns (DHs), and the involved neural cells in rats with incisional neck pain. MATERIALS AND METHODS: Male SD rats were randomly divided into control, model, Futu (LI18), Hegu-Neiguan (LI4-PC6), and Zusanli-Yanglingquan (ST36-GB34) groups. The incisional neck pain model was established by making a longitudinal incision and repeated mechanical separation along the thyroid gland region. EA (2Hz/100Hz, 1mA) was applied to LI18, LI4-PC6, ST36-GB34 separately for 30min, once at 4, 24 and 48h after incision. The local thermal pain threshold (TPT) of the focus was measured and the expression of GABA, and GABAR proteins and mRNAs detected by immunofluorescence stain and quantitative RT-PCR, respectively. RESULTS: The analgesic effect of LI18 and LI4-PC6 was superior to that of ST36-GB34 in incisional neck pain rats. Moreover, the EA stimulation of LI18 or LI4-PC6 increased the expression of GABA and GABA-Aα2 and GABA-Aß3, GABA-B1, and GABA-B2 mRNAs in spinal DHs 4h after surgery, while GABA-A and GABA-B antagonists inhibited the analgesic effect of LI18. Immunofluorescence double staining showed that GABA was expressed on astrocytes and neurons, and GABA-B expressed only on neurons. CONCLUSION: EA of both LI18 and LI4-PC6 has a good analgesic effect in incisional neck pain rats, which is closely related to their effects in upregulating the expression of GABA and its receptors in spinal DHs. The effects of LI18 and LI4-PC6 EA are obviously better that those of ST36-GB34 EA, and GABA is expressed on neurons and astrocytes.

[High Mobility Group Box 1/ CD 24 Receptor/ß-EP Signaling in "Zusanli" (ST 36) Region Contributes to Electroacupuncture Analgesia in Rats with Neuropathic Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of high mobility group box 1 (HMGB 1) and its receptor CD 24 proteins and ß-endorphin (ß-EP) content in "Zusanli" (ST 36) region in rats with chronic constriction injury (CCI), so as to explore its mechanisms underlying pain relief. METHODS: Thirty male Wistar rats were rando-mized into control, CCI model and EA groups (n＝ 10 rats in each). The neuropathic pain model was established by ligature of the left sciatic nerve to induce CCI in the model and EA groups, and sham operation was performed in rats of the control group. Paw with drawal latency (PWL, thermal pain threshold) of the bilateral hind-limbs was detected by using an algesia-detector. Eight days after CCI operation, EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 5 days. The acetylated-HMGB 1 expression was determined by immunoprecipitation, and the expression of HMGB 1 and toll like receptor 4 (TLR 4) proteins and CD 24 mRNA were detected using Western blot and fluorescent quantitative real time-PCR, respectively, and the content of ß-EP in the acupoint region was assayed by enzyme linked immunosorbent assay (ELISA). Anti-CD 24 neutralizing antibody (200 µL, 100 µg/mL) was injected into ST 36 region once daily for 3 days for verifying the involvement of HMGB 1/CD 24 signaling in EA analgesia. RESULTS: Compared with the control group, the bilateral PWL difference values in the other two groups were significantly increased (P<0.05), meaning an occurrence of hyperalgesia after CCI. In comparison with the CCI model group, the hyperalgesia in the EA group was obviously decreased (P<0.05). After CCI, the expression levels of HMGB 1 and TLR 4 proteins were considerably increased compared with those in the control group (P<0.05). After 5-times' EA, the acetylated-HMGB 1, the expression of CD 24 mRNA, and the content of ß-EP were notably up-regulated (P<0.05), and there were no obvious changes in the expression levels of HMGB 1 and TLR 4 proteins (P>0.05). After local injection of anti-CD 24 antibody, EA-induced increases of ß-EP content and reduction of thermal pain threshold were significantly suppressed (P<0.05). CONCLUSION: EA of ST 36 and GB 34 can alleviate neuropathic pain in CCI rats, which is associated with its effects in up-regulating ß-EP content, and HMGB 1 protein and CD 24 mRNA expression levels in ST 36 region. The activated HMGB 1/CD 24/ß-EP signaling contributes to EA-ST 36 induced analgesia.

[Effect of Electroacupuncture Intervention on Expression of Synaptic Plasticity-related Molecules in Amygdala in Chronic Pain-negative Affection Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of synaptic plasticity-related glutamate N-methyl-D-aspartate (NMDA) receptor subunit NR 1, gamma-aminobutyric acid (GABA) receptor subunits (Aß 2, B 1), etc. in the amygdala in chronic neuropathic pain negative affection (CNPPNA) rats, so as to reveal its mechanism underlying pain relief. METHODS: Male Wistar rats were randomized into normal control, CNPPNA model, EA, and anesthesia+EA (AEA)groups (n=14 in each group, 8 for quantitative RT-PCR and 6 for immunofluorescence staining). The CNPPNA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the hindpaw plantar skin in the pain-paired compartment. EA was applied to bilateral "Zusanli"(ST 36)and "Yanglingquan"(GB 34)for 30 min, once daily for 7 days.Thermal pain threshold (paw withdrawal latency, PWL)of the bilateral paws was measured by using a Tail-Flick Unit. The conditioned place aversion (CPA) was determined by using a CPA-paired compartment. The expression levels of GABAAß 2, GABAB 1, NMDA receptor subunit NR 1, postsynaptic density-95 protein(PSD-95), Piccolo genes in the right amygdala area were determined using quantitative RT-PCR, and the immunoactivity of metabotropic glutamate receptor subunit 1 (mGluR 1) and GABAB 2 in the basolateral amygdala (BLA) nucleus was detected using immunofluorescence staining. RESULTS: After modeling, PWL difference (PWLD) values of the model group were significantly increased (P<0.001),and the time spent in the CPA-paired compartment was considerably decreased compared with the control group (P<0.001).After EA intervention for 3 and 7 days, the PWLD levels of both EA and AEA groups were apparently decreased(P<0.05),and the time spent in the CPA-paired compartment was apparently increased in the EA and AEA groups(P<0.05),suggesting a pain relief and an improvement of the negative affection after EA intervention. Additionally, following EA, the apparently-decreased expression levels of GABAAß 2,GABAB 1,PSD-95,Piccolo genes and the reduced numbers of GABAB 2 positive cells and NMDA-NR 1 mRNA as well as mGluR 1 positive fiber numbers were remarkably increased in the EA group (P<0.05, P<0.001).The expression levels of Piccolo gene, GABAB 2 and mGluR 1 positive cells/fiber numbers were apparently lower in the AEA group than in the EA group (P<0.001). No significant differences were found between the EA and AEA groups in the PWLD, time spent in the CPA-paired compartment, and the expression levels of NMDA-NR 1, GABAAß 2, GABAB 1 and PSD-95 genes (P>0.05). CONCLUSIONS: Repeated EA stimulation of ST 36-GB 34 has a role in relieving both sensory and affection dimensions of chronic pain in CNPPNA rats, which Feb be respectively related to its effects in up-regulating the expression of GABAAß 2, GABAB 1, NMDA-NR 1, PSD-95 and Piccolo genes, and in promoting the expression of mGluR 1 and GABAB 2 proteins and Piccolo gene in the amygdala.

[Effect of Electroacupuncture Intervention on Expression of Pain Sensory and Affective Proce- ssing-related µ-opioid Receptor, etc. in the Amygdala in Chronic Neuropathy Pain Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of pain sensory and affective processing-related µ-opioid receptor (MOR), glutamatergic AMPA receptor subunit GIuA 1, extracellular signal-regulated kinase (ERK 1/2), cAMP response element binding protein(CREB) in the amygdala in chronic constrictive injury (CC) + negative affection(NA) rats, so as to reveal its mechanism underlying pain relief. METHODS: A total of 32 male Wistar rats were randomized into normal control, model, EA, and anesthesia+ EA (AEA) groups (n = 8 in each group). The neuropathic pain NA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the paw-bottom in the pain-paired compartment. EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 7 days. Thermal pain threshold (paw withdrawl latency, PWL) of the bilateral paws was measured by using a Tail-Flick Unit. The expression levels of MOR and p-CREB in the central amygdala (CeA) and those of MOR, GluA 1, p-ERK 1/2 and p-CREB in the right amygdala area were determined using immunofluorescence and Western blot, respectively. RESULTS: in comparison with the normal group, PWL difference (PWLD) values of the model group were significantly increased (P < 0.001), and the time spent in the CPA-paired. compartment was considerably decreased (P < 0.001). After EA, the PWLD levels of both EA and AEA groups were apparently decreased (P < 0.05), showing a pain relief; and the time spent in the CPA-paired compartment was apparently increased in the EA group (P < 0.05) , rather than in the AEA group (P > 0.05). Additionally , compared to the normal group, the expression level of MOR protein in the amygdala was remarkably increased (P < 0.05) and those of GIuA 1, p-ERK 2 and p-CREB proteins were apparently decreased (P < 0.05). After EA intervention for 7 days, the expression levels of these four proteins in the EA group, and those of MOR, p-ERK 2 and p-CREB in the AEA group were significantly up-regulated (P < 0.001, P < 0.01, P < 0.05). The expression level of GIuA 1 was significantly higher in the EA group than in the AEA group (P < 0.05). CONCLUSION: Repeated EA stimulation of ST 36-GB 34 has a definite effect in relieving both sensation and affection dimensions of pain in NA rats, which may be related to its effect in up-regulating the expression of GIuA 1 in the amygdala, but the effects of MOR, p-ERK 2 and p-CREB need being researched further.

[Does the Nucleus Ambiguus-Vague Nerve Mediate Anti-myocardial Ischemic Effect of Electroacupuncture of "Neiguan" (PC 6)-"Jianshi" (PC 7) in Myocardial Ischemia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Neiguan" (PC 6)-"Jianshi" (PC 7) on ischemic myocardial injury in myocardial ischemic (MI) rats, so as to explore its mechanism underlying improvement of MI. METHODS: A total of 48 male Wistar rats were randomly divided into normal, MI model, PC 6-PC 7, Sanyinjiao-Diji (SP 6-SP 8) groups (n=8 in each group for physiological experiments, n=4 in each group for immunoflorescence stain). The MI model was established by occlusion of the anterior descending branch of the left coronary artery. Electrocardiogram (ECG) of the neck-thoracic lead was recorded and the heart rate variability (HRV) analyzed by using a physiological signal collecting system (MP 150) and PowerLab software. EA (2 Hz/15 Hz, 0.5 mA) was applied to bilateral PC 6-PC 7 or SP 6-SP 8 for 30 min every time, on the 1,2,3 day after modeling. Serum creatine kinase-MB isoenzyme (CK-MB) and lactate dehydrogenase-1 (LDH 1) and endothelin (ET) contents were assayed by ELISA. The expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and c-fos proteins in the nucleus ambiguus (NA) region of the medulla oblongata was detected by immunofluo-rescence stain. RESULTS: Compared with the normal group, the ECG J-point height (1 h), serum CK-MB, LDH 1 and ET contents (1 d and 3 d), HR (1 h and 1 d) and LF/HF levels of HRV (1 h, 1 d and 3 d) and the number of c-fos positive neurons (3 d) in the NA region were significantly increased in the model group (P<0.01, P<0.05), while HF levels (1 h, 1 d and 3 d) and ECG J-point height (1 d and 3 d) were considerably decreased after MI. After EA intervention, the ECG J-point height 3 d after MI was close to zero in both PC 6-PC 7 and SP 6-SP 8 groups, being better than that (negative value) of the model group, and serum CK-MB, LDH 1 and ET contents (1 d, 3 d) of both EA groups, and HR (1 d) and LF/HF (3 d) of the PC 6-PC 7 group were obviously down-regulated (P<0.01, P<0.05), and the decreased HF 3 d post-MI and the increased number of c-fos positive neurons were significantly up-regulated in the PC 6-PC 7 group (P<0.01). No significant differences were found between the two EA groups in raising J-point height and in down-regulating serum CK-MB, LDH 1 and ET contents (P>0.05). Compared with the model group, no significant changes were found in HR 1 d post-MI of the SP 6-SP 8 group, 3 d post-MI of both EA groups, in HF 3 d post-MI of the SP 6-SP 8 group, in LF/HF 1 d post-MI of both EA groups and 3 d post-MI of the SP 6-SP 8 group, in the number of c-fos positive neurons of the SP 6-SP 8 group, and ChAT and VAChT positive neurons of both EA groups (P>0.05). CONCLUSIONS: EA intervention may improve ischemic myocardial injury in MI rats, probably by activating neurons in the NA region, and enhancing the cardiac parasympathetic tension, and balancing cardiac sympathetic/parasympathetic nerve activities, but the effect of cholinergic neurons of NA needs being studied further.