Analysis on Interrelation between Electroacupuncture-lnduced Cumulative Analgesic Effect and Hypothalamic Cholinergic Activities in Chronic Neuropathic Pain Rats

Objective： To observe the effects of repeated electroacupuncture （EA） of Zusanli （ST36）Yanglingquan （GB34） on hypothalamic acetylcholinesterase （AchE） and vesicular acetylcholine （ACh） transporter （VAChT） activities and choline acetyltransferase （CHAT） mRNA and muscarinic M1 receptor （MIR） mRNA expression in chronic constrictive injury （CCI） and/or ovariectomy （OVX） rats so as to reveal its underlying mechanism in cumulative analgesia. Methods： A total of 103 female Wistar rats were randomly divided into normal control （n=15）, CCI （n=15）, CCI＋EA2d （n=15）, CCI＋EA2W （n=15）, OVX＋CCI （n=13）, OVX＋CCI＋EA2d （n=15）, and OVX＋CCI＋EA2W groups （n=15）. CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats＇ memorylearning ability, and the thermal pain threshold （PT） of the bilateral paws was detected the next morning after EA. EA （2/15 Hz, 1 mA） was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and MIR mRNA expressions were assayed by reverse transcription-polymerase chain reaction （RT-PCR）. Results： In comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus （ARC） and supraoptic nucleus （SON） regions of CCI group, AChE activity in paraventricular nucleus （PVN）, ARC, and SON regions of OVX＋CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX＋CCI groups were down-regulated significantly （P〈0.05）. Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI＋EA2d and CCI＋EA2W groups and PVN region of CCI＋EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCi＋EA2W group were up-regulated considerably （P〈0.05）. In comparison with the OVX＋CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX＋CCI＋EA2W group were up-regulated remarkably （P〈0.05）. The effects in rats of CCI＋EA2W group were evidently superior to those of OVX＋CCI＋EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and MIR mRNA （P〈0.05）. Similar situations were found in OVX＋CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI＋EA2W and OVX＋CCI＋EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA （P〈0.05）, suggesting a reduction of EA2W effects after OVX. Conclusion： Repeated EA can significantlyup-regulate AChE and VAChT activities and ChAT mRNA and MIR mRNA expressions in the hypothalamus of CCI and OVX＋CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals＇ neuromemory ability.

Microsurgical dorsal root entry zone coagulation for chronic neuropathic pain due to spinal cord and/or cauda equina injuries

Chronic pain is a major problem for patients suffering from spinal cord injury （SCI）.The prevalence of SCI-related pain ranges from 48% to 94% depending on the method of data acquisition and type of pain considered.1 SCI-related neuropathic pain is classified into three subtypes：above-,at-,and below-lesion-level pain.After SCI,it is believed that the dorsal horn of the injured spinal cord expressed increased electrical activity.2 Thus,the neurosurgical procedures for coagulation in the dorsal root entry zone （DREZ） have proved to be most effective in chronic neuropathic pain due to SCI.The aim of this study was to describe the effect and safety ofmicrosurgical DREZ coagulation in treating chronic SCI-related neuropathic pain.

Electroacupuncture attenuates neuropathic pain after brachial plexus injury

Electroacupuncture has traditionally been used to treat pain, but its effect on pain following brachial plexus injury is still unknown. In this study, rat models of an avulsion injury to the left brachial plexus root （associated with upper-limb chronic neuropathic pain） were given electroacupuncture stimulation at bilateral Quchi （LIll）, Hegu （LI04）, Zusanli （ST36） and Yanglingquan （GB34）. After electroacupuncture therapy, chronic neuropathic pain in the rats＇ upper limbs was significantly attenuated. Immunofluorescence staining showed that the expression of β-endorphins in the arcuate nucleus was significantly increased after therapy. Thus, experimental findings indi- cate that electroacupuncture can attenuate neuropathic pain after brachial plexus injury through upregulating β-endorphin expression.

Microencapsulation improves inhibitory effects of transplanted olfactory ensheathing cells on pain after sciatic nerve injury

Olfactory bulb tissue transplantation inhibits P2X2/3 receptor-mediated neuropathic pain. However, the olfactory bulb has a complex cellular composition, and the mechanism underlying the action of purified transplanted olfactory ensheathing cells（OECs） remains unclear. In the present study, we microencapsulated OECs in alginic acid, and transplanted free and microencapsulated OECs into the region surrounding the injured sciatic nerve in rat models of chronic constriction injury. We assessed mechanical nociception in the rat models 7 and 14 days after surgery by measuring paw withdrawal threshold, and examined P2X2/3 receptor expression in L4–5 dorsal root ganglia using immunohistochemistry. Rats that received free and microencapsulated OEC transplants showed greater withdrawal thresholds than untreated model rats, and weaker P2X2/3 receptor immunoreactivity in dorsal root ganglia. At 14 days, paw withdrawal threshold was much higher in the microencapsulated OEC-treated animals. Our results confirm that microencapsulated OEC transplantation suppresses P2X2/3 receptor expression in L4–5 dorsal root ganglia in rat models of neuropathic pain and reduces allodynia, and also suggest that transplantation of microencapsulated OECs is more effective than transplantation of free OECs for the treatment of neuropathic pain.

Effects of Repeated Electroacupuncture on β-Endorphin and Adrencorticotropic Hormone Levels in the Hypothalamus and Pituitary in Rats with Chronic Pain and Ovariectomy

Objective： To explore the mechanism of electroacupuncture （EA）-induced cumulative analgesic effects on chronic pain in rats with or without ovariectomy （OVX）. Methods： A total of 110 female Wistar rats were randomized into normal control （n=10）, chronic constrictive injury （CCI, n=10）, CCI＋EA （n=30）, OVX＋CCI （n=30）, and OVX＋CCI＋EA （n=30） groups. Each of the latter 3 groups was further divided into 2 days （2 d）, 2 weeks （2 W） and 3 weeks （3 W） subgroups, respectively （n=10 in each subgroup）. The CCI pain model was established by ligature of the right sciatic nerve, and the memory impairment model duplicated by OVX. The paw withdrawal latency （PWL, pain threshold） of the bilateral footplates was detected by radiant heat irradiation, and the bilateral difference in PWL （PWLD） was used to evaluate changes in the pain reaction. Morris water maze test was conducted for evaluating the rats＇ learning-memory ability. EA was applied to bilateral Zusanli （ST36） and Yanglingquan （GB34） for 2 d, 2 W and 3 W, respectively. Pituitary and hypothalamic 13-endorphin （EP） and adrenocorticotrophic hormone （ACTH） contents were detected by immunoradioassay. Results： Compared with the CCI group, PWLD of the CCI＋EA-3 W group decreased significantly （P〈0.05）. Compared with the OVX＋CCI group, PWLD of the OVX＋CCI＋EA-3 W group was lowered considerably （P〈0.05）, but the value was markedly higher than its basal value and those of the normal control and CCI＋EA groups （P〈0.05）. In comparison with the sham-OVX group, the escape latency, swimming distance （SD） in the target quadrant and total SD were increased remarkably in the OVX group （P〈0.05）, while the number of target platform crossings was decreased significantly （P〈0.05）, suggesting an impairment of the OVX rats＇ learning-memory ability. In simple CCI rats, both β-EP and ACTH contents of the pituitary increased markedly （P〈0.05）, and those of the hypothalamus decreased obviously compared to the normal control group （P〈0.05）. After EA, pituitary and hypothalamic ACTH levels were significantly lowered at 2 d and hypothalamic ACTH and β -EP contents increased obviously at 3 W in comparison with the CCI group （P〈0.05）. In OVX＋CCI rats, following EA, pituitary β -EP contents at 2 d, 2 W and 3 W, and hypothalamic β-EP and ACTH contents at 2 W and hypothalamic ACTH levels at 3 W increased significantly （P〈O.05）, but hypothalamic β-EP level at 3W decreased markedly （P〈0.05）. The effects of repeated EA in lowering pituitary ACTH and raising hypothalamic β-EP and ACTH levels disappeared after OVX＋CCI. Conclusions： Repeated EA has a cumulative analgesic effect, which is closely associated with its effects in regulating pituitary and hypothalamic β-EP and ACTH levels. OVX may weaken the analgesic effect of EA by affecting hypothalamic-pituitary axis activity.

Correlation between the cumulative analgesic effect of electroacupuncture intervention and synaptic plasticity of hypothalamic paraventricular nucleus neurons in rats with sciatica

In the present study, a rat model of chronic neuropathic pain was established by ligation of the sciatic nerve and a model of learning and memory impairment was established by ovariectomy to investigate the analgesic effect of repeated electroacupuncture stimulation at bilateral Zusanfi （ST36） and Yanglingquan （GB34）. In addition, associated synaptic changes in neurons in the paraventricular nucleus of the hypothalamus were examined. Results indicate that the thermal pain threshold （paw withdrawal latency） was significantly increased in rats subjected to 2-week electroacupuncture intervention compared with 2-day electroacupuncture, but the analgesic effect was weakened remarkably in ovariectomized rats with chronic constrictive injury. 2-week electroacupuncture intervention substantially reversed the chronic constrictive injury-induced increase in the synaptic cleft width and thinning of the postsynaptic density. These findings indicate that repeated electroacupuncture at bilateral Zusanfi and Yanglingquan has a cumulative analgesic effect and can effectively relieve chronic neuropathic pain by remodeling the synaptic structure of the hypothalamic paraventricular nucleus.

KCNQ Channels in the Mesolimbic Reward Circuit Regulate Nociception in Chronic Pain in Mice

Mesocorticolimbic dopaminergic(DA) neurons have been implicated in regulating nociception in chronic pain, yet the mechanisms are barely understood. Here, we found that chronic constructive injury(CCI) in mice increased the firing activity and decreased the KCNQ channel-mediated M-currents in ventral tegmental area(VTA) DA neurons projecting to the nucleus accumbens(NAc). Chemogenetic inhibition of the VTA-to-NAc DA neurons alleviated CCI-induced thermal nociception.Opposite changes in the firing activity and M-currents were recorded in VTA DA neurons projecting to the medial prefrontal cortex(mPFC) but did not affect nociception. In addition, intra-VTA injection of retigabine, a KCNQ opener, while reversing the changes of the VTA-to-NAc DA neurons, alleviated CCI-induced nociception, and this was abolished by injecting exogenous BDNF into the NAc.Taken together, these findings highlight a vital role of KCNQ channel-mediated modulation of mesolimbic DA activity in regulating thermal nociception in the chronic pain state.

The cumulative analgesic effect of repeated electroacupuncture involves synaptic remodeling in the hippocampal CA3 region

In the present study, we examined the analgesic effect of repeated electroacupuncture at bilateral Zusanfi （ST36） and Yanglingquan （GB34） once a day for 14 consecutive days in a rat model of chronic sciatic nerve constriction injury-induced neuropathic pain. In addition, concomitant changes in calcium/calmodulin-dependent protein kinase II expression and synaptic ultrastructure of neurons in the hippocampal CA3 region were examined. The thermal pain threshold （paw withdrawal latency） was increased significantly in both groups at 2 weeks after electroacupuncture intervention compared with 2 days of electroacupuncture. In ovariectomized rats with chronic constriction injury, the analgesic effect was significantly reduced. Electroacupuncture for 2 weeks significantly diminished the injury-induced increase in synaptJc cleft width and thinning of the postsynaptJc density, and it significantly suppressed the down-regulation of intracellular calcium/ calmodulin-dependent protein kinase II expression in the hippocampal CA3 region. Repeated electroacupuncture intervention had a cumulative analgesic effect on injury-induced neuropathic pain reactions, and it led to synaptic remodeling of hippocampal neurons and upregulated calcium/calmodulin-dependent protein kJnase II expression in the hippocampal CA3 region.