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Objective: To observe the effect of electroacupuncture (EA) on changes of expression of L-Arg transporter 2 (CAT-2) mRNA and nitric oxide synthase (iNOS) mRNA and protein and contents of NO and cGMP of L 4 -L 6 segments of spinal cord in rats with spared nerve injury (SNI), so as to reveal its mechanism underlying reducing neuropathic pain ., Methods: A total of 120 male SD rats were randomly divided into sham operation, model, EA and NOS inhibitor (N omega-Nitro-L-arginine methyl ester hydrochloride, L-NAME) groups, with 30 rats in each group. The neuropathic pain model was established by ligating and cutting the tibial nerve and the common peroneal nerve. EA (2 Hz, 1-3 mA) was applied to "Weizhong" (BL40) and "Huantiao" (GB30)on the damaged hindlimb for 30 min, once daily from day 11 to 17 after SNI. Rats of the L-NAME group received i.p. of L-NAME (60 mg·kg -1 ·d -1 ) for 7 consecutive days. The mechanical pain threshold (PT) was determined before and 10 and 16 d after SNI, respectively. The expression le-vels of CAT-2 mRNA and iNOS mRNA, and iNOS protein in the L 4 -L 6 segments of the spinal cord were detected by using reverse transcription - polymerase chain reaction (RT-PCR) and Western blot, respectively, and the contents of NO and cGMP of L 4 -L 6 assayed using nitrate/nitrite reductase method and radioimmunoassay, respectively ., Results: After modeling, the PT was significantly decreased on day 10 and 16 after SNI in comparison with the sham operation group and their own baseline data of pre-operation in each group ( P < 0.01), and remarkably increased in the EA and L-NAME groups relevant to the model group on day 16 ( P < 0.01, P < 0.05). Compared with the sham operation group, the expression levels of CAT-2 mRNA and iNOS mRNA and protein, as well as the contents of NO 2 － /NO 3 － and cGMP were signi-ficantly up-regulated in the model group ( P < 0.05, P < 0.01). Following EA intervention, the levels of CAT-2 mRNA and iNOS mRNA and iNOS protein, and NO 2 － /NO 3 － and cGMP contents were all reversed in both EA and L-NAME groups ( P < 0.05, P < 0.01). The effect of EA was significantly superior to that of L-NAME in raising the PT on day 16 after SNI ( P < 0.05), but obviously inferior to that of L-NAME in down-regulating the expression of CAT-2 mRNA and iNOS mRNA and protein ( P < 0.05). No significant differences were found between the EA and L-NAME groups in down-regulating NO 2 － /NO 3 － andcGMP contents ( P > 0.05)., Conclusion: EA intervention can effectively relieve neuropathic pain in SNI rats, which may be closely related to its function in suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord.

Objective: We have demonstrated that electroacupuncture (EA) of "Weizhong" (BL 40) and "Huantiao" (GB 30) could evidently relieve mechanical hyperalgesia in spared nerve injury (SNI) rats. The present study was designed to observe the effect of EA on levels of cAMP, and protein kinase A (PKA) and cAMP response element binding protein (CREB) in the lumbar spinal cord of the same pain model rats, so as to explore its mechanisms underlying improvement of neuropathic pain., Methods: One hundred male SD rats were randomly divided into 5 groups: sham operation, model, EA, AP-5 and L-NAME groups ( n ＝20 in each group). The sham operation group underwent a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right BL 40 and GB 30 for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg•kg －1 •d －1 ) and L-NAME (a non-selective antagonist for nitric oxide synthase, NOS, 60 mg•kg －1 •d －1 ) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, the cAMP content of spinal cord (L 4－L 6) was determined by radioimmunoassay, and the expression of PKA, p-PKA and CREB proteins of spinal cord (L 4－L 6) was detected by Western blot., Results: The content of cAMP and the expression levels of PKA, p-PKA and CREB were significantly higher in the model group than in the sham operation group ( P <0.01), and considerably lower in the EA group than in the model group ( P <0.01, P <0.05). Compared to the model group, the expression levels of PKA and p-PKA were significantly decreased in the AP-5 and L-NAME groups ( P <0.01, P <0.05). Compared to the EA group, the content of cAMP and the expression levels of CREB were significantly higher ( P <0.05), and the expression of p-PKA was significantly lower in the AP-5 and L-NAME groups ( P <0.05)．., Conclusion: EA intervention-induced down-regulation of cAMP, PKA and CREB levels in the lumbar spinal cord may contribute to its analgesic effect in neuropathic pain rats, suggesting an involvement of reduction of cAMP/PKA/CREB signaling of spinal cord in EA analgesia.

Objective: To observe the changes of intracellular calcium ([Ca 2＋ ]i) concentration and expression of calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) in spinal dorsal horn neurons of spared nerve injury (SNI) rats, so as to explore its mechanisms underlying improvement of neuropathic pain., Methods: One hundred and ten SD rats were randomly divided into 5 groups: sham control, model, EA, AP-5 and L-NAME groups. The sham group underwent only a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right "Weizhong" (BL 40) and "Huantiao" (GB 30) for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg·kg －1 ·d －1 ) and L-NAME (a non-selective antagonist for nitric oxide synthase [NOS], 60 mg·kg －1 ·d －1 ) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, and the calcium fluorescence intensity (shown by Fluo-3/AM calcium fluorescence indicator) of the superficial layer of the lumbar spinal cord (L 4－L 6) was measured by immunohistochemical staining and the expression of spinal cord (L 4－L 6) CaMK Ⅱ protein was detected by Western blot (WB)., Results: After modeling, the mechanical pain threshold was significantly decreased on day 10 and 16 after operation in comparison with the sham operation group and baseline data of pre-operation in each group ( P <0.01), and remarkably increased in the EA, AP-5 and L-NAME groups relevant to the model group on day 16 ( P <0.01, P <0.05), while the effect of EA was significantly superior to that of AP-5 and L-NAME groups ( P <0.05), suggesting a reduction of EA analgesia after administration of AP-5 and L-NAME. The concentration of intracellular [Ca 2＋ ]i was significantly higher in the model group than in the sham group, and considerably lower in the EA, AP-5 and L-NAME groups than in the model group ( P <0.01, P <0.05). Moreover, the expression level of CaMKⅡ shown by WB and immunohistochemical staining was significantly higher in the model group than in the sham group ( P <0.05) and obviously lower in the EA group (not the AP-5 and L-NAME groups) than in the model group on day 16 after the intervention ( P <0.05). It suggests an involvement of glutamate NMDA receptor and NMDAR-NOS/NO signaling in the analgesic effect and CaMKⅡ expression down-regulation of EA., Conclusions: EA can ease pain in rats with neuropathic pain, which is closely related to its effect in reducing the calcium concentration and the expression of CaMKⅡ in the lumbar spinal cord, possibly mediated by glutamate NMDA receptor and NMDAR-NOS/NO signaling.

Unlabelled: OBJECTIVE; To observe the changes of N-methyl-D-aspartic acid (NMDA) receptor expression of spinal cord after electroacupuncture (EA) intervention in rats with chronic constrictive injury (CCI) of the sciatic nerve so as to reveal the mechanism underlying improvement of neuropathic pain., Methods: Sixty male SD rats were randomly divided into sham operation (sham) group, CCI model group and EA group (n = 20). CCI model was established by ligature of the right sciatic nerve with a piece of surgical chromic suture. For rats of the sham group, the right sciatic nerve was just isolated without ligature. The rats of the EA group were given with EA stimulation of "Weizhong" (BL 40) and "Huantiao" (GB 30) on the injured side at a frequency of 2 Hz, electric current of 1-3 mA for 30 min (increasing 1 mA every 10 min). The treatment was conducted once a day from the 11th day to the 20th day after modeling. NMDA receptor 2 B subunit (NR 2 B) protein and mRNA expression levels in the spinal cord were determined by immunohistochemistry, Western blot and reverse transcription (RT)-polymerase chain reaction (PCR), respectively, and spinal NMDA receptor subunit 1 (NR 1) protein and mRNA expression levels were measured by Western blot and RT-PCR, respectively., Results: In comparison with the sham group, NR 1 protein and mRNA expression levels of the model group in the spinal cord were considerably upregulated after CCI (P < 0.01, P < 0.05). In comparison with the model group NR 1 protein and mRNA expression levels of the EA group in the spinal cord were evidently down-regulated (P < 0.05). No significant changes of NR 2 B protein and mRNA expression after CCI and EA stimulation were found after immunohistochemistry, Western blot and RT-PCR measurements (P > 0.05)., Conclusion: EA intervention is effective in alleviating neuropathic pain in CCI rats, which may be closely related to its effects in lowering functional activity of NR 1 protein and mRNA in the spinal cord.

Objective: To observe the effect of electroacupuncture (EA) on expression of neuronal nitric oxide synthase (nNOS) protein and mRNA in the spinal cord in rats with chronic constrictive injury (CCI) of the sciatic nerve so as to explore its mechanisms underlying pain relief., Methods: Thirty-six male SD rats were randomly divided into sham operation (control) group (n = 12), CCI model group (n = 12) and EA group (n = 12). EA was applied to bilateral "Weizhong" (BL 40) and "Huan-tiao" (GB 30) on the injured side for 30 min, once a day for 7 days (from the 11th day on after CCI). The mechanical and thermal pain thresholds were measured before and after CCI. Spinal nNOS protein and mRNA expression levels were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively., Results: In comparison with the control group, both mechanical and thermal pain thresholds were significantly lower in the model group (P < 0.01). Compared with the model group, the two types of pain threshold were increased considerably on the 16th day in the EA group (P < 0.01). In comparison with the control group, the spinal nNOS protein and mRNA expression levels were increased significantly in the model group (P < 0.05, P < 0.01). In comparison with the model group, both nNOS protein and mRNA expression levels in the spinal cord were down-regulated obviously in the EA group (P < 0.05)., Conclusion: Electroacupuncture can alleviate neuropathic pain in CCI rats, which is closely related to its effect in down-regulating the expression of nNOS protein and mRNA in the spinal cord.

Objective: To observe the changes of amino acid neurotransmitter contents in the lumbar spinal cord after electroacupuncture (EA) in rats with neuropathic pain so as to study its spinal mechanism underlying pain relief., Methods: Forty SD rats were randomly divided into control, sham surgery, model and EA groups (n=10). Chronic constrictive injury (CCI) (neuropathic pain) model was duplicated by ligature of the right sciatic nerve with a piece of catgut. EA (1-3 mA, 2 Hz) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the injured side for 30 minutes, once a day for 7 days. The mechanical and thermal pain thresholds were measured before and after CCI, and after EA intervention. Concentrations of glutamate (Glu), aspartate (Asp), glutamine (Gln), gamma-aminobutyric acid (GABA), Glycine (Gly) and taurine (Tau) in the lumbar spinal cord (L4-6) were detected by O-phthaladehyde derivatization + high performance liquid chromatography (HPLC)., Results: Compared with the control group, both the mechanical and thermal pain thresholds of the model group were decreased significantly on day 10 and day 16 after CCI (P < 0.01). Compared with the model group, the mechanical and thermal pain thresholds in the EA group on day 16 after CCI were upregulated strikingly (P < 0.01). In comparison with the control group, Glu, Asp, Gin and GABA levels in the lumbar spinal cord were significantly higher in the model group (P < 0.05, P < 0.01), while Gly and Tau levels in the lumbar spinal cord were markedly lower in the model group (P < 0.05). In comparison with the model group, spinal Glu, Asp, and Gin contents were downregulated significantly in the EA group (P < 0.01), while Gly, GABA and Tau levels were upregulated obviously (P < 0.01, P < 0.05). No significant differences were found between the control and sham surgery groups in the mechanical and thermal pain thresholds, and in the spinal Glu, Asp, GIn, GABA, Gly and Tau levels (P > 0.05)., Conclusion: EA of GB 30 and BL 40 may alleviate neuropathic pain in CCI rats, which is closely with its effects in reducing the release of excitatory amino acids and promoting the release of inhibitory amino acid neurotransmitters.

Objective: To observe the effect of electroacupuncture (EA) on the expression of ionotropic glutamate receptor (iGluR) subunits and their mRNAs in the lumbar segments of spinal cord in rats with neuropathic pain, so as to explore its underlying mechanism in relieving spinal hyperalgesia., Methods: Thirty SD rats were randomly divided into control, model, and EA groups, with 10 rats in each. The spared nerve injury (SNI) model was established by ligature of the sural nerve after cutting off the common peroneal nerve and anterior tibial nerve. EA (2 Hz, 1 mA) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) for 30 min, once daily for 7 days. Mechanical pain threshold was detected before and after modeling and before and after EA treatment. The expression levels of N-methyl-d-aspartic acid (NMDA) receptor subunits NR1 and NR 2 B,and AMPA receptor subunit GluR 1 of iGluR and their genes were assayed by Western blot and reverse transcription polymerase chain reaction (RT-PCR) separately., Results: In comparison with control group, the mechanical pain thresholds were decreased significantly on day 2, 7 and day 14 following modeling in the model group (P < 0.05, P < 0.01). While compared with the model group, the pain threshold was increased considerably on day 14 in the EA group (P < 0.01). Compared with the control group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the model group were increased significantly (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the model group increased slightly (P > 0.05). In comparison with the model group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the EA group were downregulated remarkably (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the EA group down-regulated slightly (P > 0.05)., Conclusion: EA can significantly suppress pain reaction in rats with neuropathic pain probably through down-regulating the expression of lumbar spinal cord NR 2 B protein and NR 2 B mRNA.

Objective: To observe the effect of electroacupuncture (EA) of "Huantiao" (GB 30) and "Weizhong" (BL 40) on the mechanical pain threshold and the long-term potentiation (LTP) of the spinal dorsal horn in rats with neuropathic pain., Methods: Thirty SD rats were randomized into control (sham-operation), model and electroacupuncture (EA) groups, with 10 cases in each. The neuropathic pain model was established by ligature and transaction of the popliteal nerve and common peroneal nerve. EA (2 Hz, 1 mA) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side for 30 min, once daily for 7 days. The mechanical pain threshold of the paw was detected before and after modeling and EA. LTP of C-fiber stimulation (2 mA, 0.5 ms, 100 Hz, interval 10 s, 4 strains) evoked field potentials in spinal dorsal horn (L4/L5) was recorded in rats under anesthesia., Results: Compared with control group, the pain threshold of model group reduced significantly from the 1st day on after ligature and transaction of the nerve (P<0.01), while 14 days after EA, it elevated evidently in comparison with model group (P<0.01), suggesting an increase of the mechanical pain threshold after EA. Comparison of the variance rates of C-fiber induced LTP of the field potential among the 3 groups showed that the variance rate of EA group was significantly lower than that of model group (P<0.01). Self-comparison of EA group showed that after EA, the variance rate of LTP reduced significantly (P<0.01)., Conclusion: EA of "Huantiao" (GB 30) and "Weizhong" (BL 40) can significantly relieve neuropathic pain in rats with nerve injury, and inhibit C-fiber high-frequency-stimulation induced LTP due to central sensitization induced abnormality synaptic transmission.

Objective: To observe the effect of electroacupuncture (EA) on pain threshold and contents of excitatory amino acids (EAA) in dorsal root ganglia (DRG) and spinal cord in rats with neuropathic pain., Methods: Fifty SD rats were randomly divided into control (C),model (M), sham-model (SM), EA, and sham-EA groups, with 10 cases in each. Neuropathic pain (spared nerve injury, SNI) model was established by cutting off the right common peroneal nerve and proso-tibial nerve (with the sural nerve reserved intact). Before and after surgery, the mechanical pain threshold (MPT) and thermal pain threshold (TPT) were measured respectively on the injured side under consciousness state. EA (2 Hz, 1-3 mA, adding 1 mA/10 min) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side for 30 min. For rats of sham-EA group, filiform needles were inserted into GB30 and BL40 simply without manipulation or electrical stimulation. The treatment was given once daily for 7 days. On the 15th day, the rats were sacrificed for sampling right L4-L6 DRG and spinal cord. The contents of neurotransmitters, glutamate (Glu) and aspartic acid (Asp) in DRG and spinal cord were detected with high performance liquid chromatography (HPLC). Micro-dialysis technique was used to collect the dialysate from the spinal cord, homogenated for measuring EAA by HPLC., Results: In comparison with control group, after SNI, MPT decreased significantly from the 1st day on in model group. Compared with model group, on the 15th day, MPT increased significantly in both EA and sham-EA groups (P < 0.05, 0.01),but MPT of sham-EA was significantly lower than that of EA group (P < 0.05). No significant changes were found in TPT in 5 groups (P > 0.05). It suggested that sham EA still had an analgesic effect in spite of being lower than that of true EA. Compared with control group, the contents of Glu and Asp in the spinal cord tissue and micro-dialysate in model group increased significantly after SNI (P < 0.01). In comparison with model group, the contents of Glu in DRG and spinal cord tissue and micro-dialysate, and Asp in the spinal cord tissue and micro-dialysate in both sham-EA and EA groups decreased considerably (P < 0.05, 0.01). Dialysate Glu and spinal cord tissue Asp contents in EA were remarkably lower than those in sham-EA group (P < 0.01, 0.05)., Conclusion: EA has a significant analgesic effect in SNI rats, which is closely related with its effect in inhibiting the release of Glu and Asp from dorsal horns of the spinal cord and lowering contents of EAAs.