Repeated electroacupuncture treatment attenuated hyperalgesia through suppression of spinal glial activation in chronic neuropathic pain rats.

BACKGROUND: Cumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown. METHODS: A total of 120 male Wistar rats were used in the present study. The neuropathic pain model was established by chronic constrictive injury (CCI) of the sciatic nerve. The rats were randomly divided into sham group, CCI group, and sham CCI + EA group, and CCI + EA group. EA was applied to bilateral Zusanli (ST36)-Yanlingquan (GB34). The mechanical (both time and force responses) and thermal pain thresholds (PTs) of the bilateral hind-paws were measured. The number of microgliacytes and activity of astrocytes in the dorsal horns (DHs) of lumbar spinal cord (L4-5) were examined by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) protein was detected by western blot. RESULTS: Following CCI, both mechanical and thermal PTs of the ipsilateral hind-paw were significantly decreased beginning from the 3rd day after surgery (P < 0.05), and the mechanical PT of the contralateral hind-paw was considerably decreased from the 6th day on after surgery (P < 0.05). CCI also significantly upregulated the number of Iba-1 labeled microgliacytes and the fluorescence intensity of glial fibrillary acidic protein (GFAP) -labeled astrocyte in the superficial laminae of DHs on bilateral sides (P < 0.05). After repeated EA, the mechanical and thermal PTs at bilateral hind-paws were significantly relieved (P < 0.05). The increased of number of microgliacytes was markedly suppressed by 2 days' EA intervention, and the average fluorescence intensity was suppressed by 2 weeks' EA. The expression of GFAP protein were down-regulated by 1 and 2 weeks' EA treatment, respectively (P < 0.05). CONCLUSIONS: Repeated EA can relieve neuropathic pain and mirror-image pain in chronic neuropathic pain rats, which is probably associated with its effect in downregulating glial cell activation of the lumbar spinal cord, the microgliacyte first and astrocyte later.

[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.

Involvement of hippocampal acetylcholinergic receptors in electroacupuncture analgesia in neuropathic pain rats.

BACKGROUND: Cumulating evidence has shown a close correlation between electroacupuncture stimulation (EAS) frequency-specific analgesic effect and central opioid peptides. However, the actions of hippocampal acetylcholinergic receptors have not been determined. This study aims to observe the effect of different frequencies of EAS on the expression of hippocampal muscarinic and nicotinic acetylcholinergic receptors (mAChRs, nAChRs) in neuropathic pain rats for revealing their relationship. METHODS: Forty male Wistar rats were randomly and equally divided into sham, CCI model, 2, 2/15 and 100 HzEA groups. The neuropathic pain model was established by ligature of the left sciatic nerve to induce chronic constriction injury (CCI). EAS was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 30 min, once daily for 14 days except weekends. The mechanical pain thresholds (withdrawal latencies, PWLs) of bilateral hindpaws were measured. The expression levels of hippocampal M1 and M2 mAChR, and α4 and ß2 nAChR genes and proteins were detected by quantitative RT-PCR and Western blot, separately. The involvement of mAChR and nAChR in the analgesic effect of EAS was confirmed by intra-hippocampal microinjection of M1mAChR antagonist (Pirenzepine) and α4ß2 nAChR antagonist (dihydro-beta-erythroidine) respectively. RESULTS: Following EAS, the CCI-induced increase of difference values of bilateral PWLs on day 6 and 14 was significantly reduced (P < 0.05), with 2/15 Hz being greater than 100 Hz EAS on day 14 (P < 0.05). After 2 weeks' EAS, the decreased expression levels of M1 mAChR mRNA of both 2 and 2/15 Hz groups and M1 mAChR protein of the three EAS groups, α4 AChR mRNA of the 2/15 Hz group and ß2 nAChR protein of the three EAS groups were considerably increased (P < 0.05), suggesting an involvement of M1 mAChR and ß2 nAChR proteins in EAS-induced pain relief. No significant changes were found in the expression of M2 mAChR mRNA and protein, α4 nAChR protein and ß2 nAChR mRNA after CCI and EAS (P > 0.05). The analgesic effect of EAS was abolished by intra-hippocampal microinjection of M1mAChR and α4ß2 nAChR antagonists respectively. CONCLUSIONS: EAS of ST36-GB34 produces a cumulative analgesic effect in neuropathic pain rats, which is frequency-dependent and probably mediated by hippocampal M1 mAChR and ß2 nAChR proteins.

[Effect of electroacupuncture intervention on expression of pain sensory and affection processing related corticotropin-releasing factor receptor mRNA, etc. in the amygdala in neuropathic pain and negative affection rats].

OBJECTIVE: To observe the effect of electroacupuncture .(EA) stimulation of "Zusanli" (ST 36)-"Yang- lingquan" (GB 34) on expression of pain sensory and affection processing-related corticotropin-releasing factor (CRF) receptor, glutamatergic NMDA receptor and GABA receptor subtype genes in the amygdala in chronic constrictive injury (CCI) of the sciatic nerve rats, so as to reveal its mechanism underlying pain relief. METHODS: Experiments were separately performed in 36 male Wistar rats which were randomized into normal control, CCI model and EA + CCI; normal control, CCI + negative affection (NA) model and CCl+ NA+ EA groups (n =6 in each group). Neuropathic pain model was established by ligature of the left sciatic nerve, and NA model established by ligation of the left sciatic nerve and repeated skin stimulation (acupuncture needle pricking + direct current stimulation) of the paw-bottom, once daily for 3 days. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral ST 36-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 37360. Expression levels of CRF-1 R, CRF-2 R, NR 2 A,NR 2 B,GABAaR and GABAcR genes in the amygdala were determined using quantitative RT-PCR. RESULTS: In comparison with the normal control groups, PWL difference (PWLD) values of the bilateral paws of CCIl model and CCI+NA model groups were significantly increased (P<0. 05). In comparison with the model group, following 7 days' EA stimulation, PWLD were considerably decreased (P<0. 05), showing a pain relief. RT-PCR results indicated that compared to the normal control group, the expression levels of CRF-1 R, CRF-2 R, NR 2 A and NR 2 B genes were apparently increased in the CCI model group (P<0. 01, P<0. 001), and those of CRF-1 R, CRF-2 R, NR 2 A, NR 2 B, GABAaR and GABAcR genes were remarkably down-regulated in the CCI + NA model group (P<0. 05, P<0. 01, P<0. 001). After EA intervention for 7 days, CRF-2 R, NR 2 A and NR 2 B were significantly down-regulated in the CCI + EA group, and CRF-1 R, CRF-2 R, NR 2 B,GABAaR and GABAcR genes were obviously up-regulated in the CCI + NA + EA group (P<0. 01, P<0. 001). CONCLUSION: Repeated EA stimulation of ST 36-GB 34 has a definite analgesic effect in neuropathic pain and negative affection rats, which may be respectively related to its effects in down-regulating expression of CRF-2 R, NR 2 A and NR 2 B genes, and up-regulating expression of CRF-1 R, CRF-2 R, NR 2 B,GABAaR and GABAcR genes in the amyg- dala.