| BACKGROUNDPain is an unpleasant subjective feeling and emotional experience associated with tissue damage or potential damage. Pain has a strong emotional component, and a mutual influence exists between pain sension and negative mood. Pressure can suppress or aggravate the pain severity, and anxiety and depression are often associated with intensive pain feelings, especially under chronic pain. Chronic pain still exists even original lesion has disappeared. The composite mechanism of pain, fear, anxiety, depression, cognitive and behavior is still a mystery. Functional MRI results show that the individuals without history of chronic pain are different from patients with chronic low back pain and pain fear escape behavior in the amygdala activation, at the same time, the amygdala and the anterior cingulate link is also different. The amygdala is also involved in conditioned fear of emotional processing, the pain memories and pain motivations. Researches on the role of amygdala in pain processing provide us with a better understanding about the underlying mechanism of pain control. Some related researches shows that the endogenous opioid system is involved in the stress reaction and emotional reaction, and the activation of mu opioid receptors is associated with the reduction of pain feeling and emotional rating. AMPA subtypes Glutamate Receptors mediate fast excitatory synaptic transmission in the central nervous system and are the main excitatory receptors of the postsynaptic membrane of neurons, and the GluAl subtype is involved in regulating depression behavior response. Synaptophysin exists in almost all of the nerve terminals of synaptic vesicles, and is closely related to the neurotransmitter release and synaptic plasticity changes. Extracellular signal-regulated Kinase(ERK 1/2) is an important member of the Mitogen-activated protein Kinase (MAPK) family, and responses to the release of neurotransmitters, peptide hormone, growth hormone, etc. It also mediate Kinase RSK2 (ribosomal S6 kinases, downstream of ERK Kinase) activities, through cyclic adenosine monophosphate response element-binding protein(CREB) phosphorylation, and then launches a variety of functional proteins. This process widely exists in the Long-term potentiation and the formation of long-term memory. Related studies have shown that ERK-CREB pathway not only participate in the dimension of pain feeling but also involve in the dimension of pain emotion.Chronic pain, is not only associated with pain sensation, but also with negative emotions and cognitive disorders, and negative emotions play a promoting effect on generating and maintaining chronic pain. The usual treatment of chronic pain is surgery and drug therapy, but the curative effect is not very satisfactory and more often associated with side effects. Acupuncture therapy has a long history of thousands of years in China, and a lot of clinical trials showed that acupuncture could relieve pain sensory and the accompanyied negative emotions. Experimental studies on the animal model of chronic pain displayed that acupuncture intervention has a good efficacy in easing pain feeling, however, there are few reports on its mechanisms underlying improvement pain emotion because of the need of simulating clinical symptoms on animal models and the limit of detection methodsOur previous studies on Chronic Neuropathy pain model indicated that acupuncture could improve behavioral responses to the pain feeling component and emotion component,and the underline mechanism is likely through reducing the CCI rats’N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B and corticotropin-releasing factor receptor-2 (CRF-2R) gene expression, raising the neuralgia negative emotion related CRF-2R, NR2B, GABA receptor GABAaR, GABAcR gene expression in amygdale. Its specific mechanism is complex, so needs further research. In present study, The neuropathic pain negative affection (NA)model was established by ligation of the left sciatic nerve and repeated electric stimulation of the paw-bottom in pain-paired compartment of a place conditioning apparatus. The analgesic effect of EA stimulation of ST 36-GB 34 in relieving both sensation and affection dimensions of pain in NA rats were observed and the related protein expression levels of glutamate AMPA receptor subtypes GluAl, mu opioid receptor, Synaptophysin, phosphorylated extracellular signal regulating kinase (p-ERK1/2), phosphorylation cAMP response element binding protein (p-CREB)in the amygdale were detected by using immunofluorescence staining and western blotting technique separately. In addition, we applied mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thra-mide (CTOP, a selective mu opioid receptor antagonist), GluAl antagonists N-[3-[(4-[(3Aminoprop yl)amino]butyl]amino]propyl]-1-naphthaleneacetamide trihydrochlorid e (NASPM) into the amygdala to verify the results of EA in relieving the pain sensation and pain affection dimentions, respectively, so as to provide theoretical basis for acupuncture treatment of chronic pain.Materials and MethodsPart OneWistar male rats(230g-260g) were randomized into normal control, model, EA, and anesthesia+EA(AEA) groups (n=8 in each group). Except the Normal group, the neuropathic pain negative affection models were established by ligation of the left sciatic nerve(chronic constrictive injury, CCI) and repeated electrical stimulation of the paw-bottom in pain-paired compartment(once daily for 3 days). EA (1mA,2/15Hz) was applied to bilateral ST36 and GB34 for 1mA,2/15Hz,30min, once daily for 7 days. The dimension of pain felling was measured by Thermal pain threshold (paw withdrawl latency, PWL) of the bilateral paws, and the dimension of pain emotion was measured by testing the time spent in pain-paired compartment of place conditioning apparatus. The expression levels of MOR and p-CREB in the amygdale and those of MOR, GluA1, synaptophysin, p-ERK1/2 and p-CREB in the right amygdale area were determined using immunof luorescence (n=6 in each group) and Western blot (n=6 in each group), respectively.Part TwoIn order to verify the effects of EA in relieving pain filling and NA dimensions,30 Wistar male rats were randomized into normal control, NA model, EA+normal saline (NS), EA+CTOP(MOR antagonist), EA+NASPM (GluA1 antagonist) groups (n=6 in each group, other 10 rats were excluded due to desquamation of the implanted cannula). Except the Normal group, the neuropathic pain negative affection model was established by using the same methods mentioned above. In addition to the normal and NA model groups, the rest rats received bilateral implantation of stainless cannulae in the amygdala (P2.5mm, L4.5mm, H7.7mm) on a stereotaxic instrument in accordance with the Paxinos and Watson Atlas.Bilateral microinjection of normal saline (0.25 μL), opioid mu receptor antagonist (CTOP) (27μg/uL,0.25μL), GluA1 antagonists (NASPM) (10μg/uL,0.25 u L) were conducted before each EA stimulation, separately. After the microinjection, EA (1mA,2/15Hz) was applied to bilateral ST 36 and GB34 for 30min (by placing the rat in an open animal box without restraint), once daily for 5 days. Then, the thermal pain thresholds of the bilateral paws and the time spent in the pain-paired compartment were detected on day 2 and 5 after the microinjection.Results1 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), suggesting a decrease of pain threshold and a formation of conditioned place aversion (CPA). After EA, 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 group (P<0.06), rather than in the AEA group (P>0.05), indicating a pain relief and an improvement of CPA after EA.2 Compared to the normal control group, the expression level of MOR protein of the amygdala was remarkably increased (P<0.05) and those of GluAl, synaptophysin, p-ERK2 and p-CREB proteins were apparently decreased in the model group (P<0.05). After EA intervention for 7 days, the expression levels of these five proteins in the EA group, and those of MOR, p-ERK2 and p-CREB in the AEA group were significantly up-regulated (P<0.001, P<0.01, P<0.05). The expression levels of GluAl and synaptophysin were significantly higher in the EA group than in the AEA group (P<0.05). It suggests an involvement of amygdaloid MOR, p-ERK2 and p-CREB proteins in the pain sensation relief, and an involvement of GluAl and synaptophysin in pain NA reduction.3 After intra-amydaloid mincroinjection of normal saline and NASPM (an antagonist of GluAl), the increased PWLD in neuropathic NA rats were significantly down-regulated (P<0.001) on day 5 after EA of Zusanli (ST36)-Yanglingquan (GB34) in the EA+NS and EA+NASM groups (P<0.01), rather than in the EA+CTOP (an antagonist of MOR) group (P>0.05), suggesting an involvement of MOR (not GluAl) in pain relief. Moreover, after intra-amygdaloid microinjection, the decreased time spent in the CPA-paired compartment was apparently increased after EA for 2 and 5 days in both the EA+NS and EA+CTOP groups (P<0.05, P<0.01, P<0.001), rather than in the EA+NASPM group (P>0.05), indicating an involvement of amygdaloid GluAl in NA relief of EA intervention. These data verified that EA induced inhibition of pain sensory dimension is mediated by amygdloid μ-opioid receptors and EA-induced reduction of pain affective response is mediated by amygdaloid GluAl.ConclusionThe present study demonstrates that EA significantly inhibits the sensory and affective dimensions of pain in Chronic Neuropathy pain Rats. EA benefit the sensory and affective dimension of pain through Amygdala μ-opioid receptors and GluAl Receptors respectively. Additionally, the up-regulate of amygdala ERK2 and p-CREB may also be involved in improving sensory component of pain, and rise in synaptic protein expression may be involved in improving the affection component of chronic pain, which need further validation. |
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