| BackgroundAcupuncturology is an important part of Chinese medicine. Acupuncture therapy has a unique advantage in treating various kinds of pain problems. A recent survey showed that the best clinical indications of acupuncture therapy for pain are lower back pain, headache, cervical spondylosis, periarthritis of shoulder, dysmenorrhea, stomachache, etc. As we know that, patients suffering from chronic pain often have some symptoms of anxiety, insomnia, irritability, depression, etc. The pain sensation and affection components are mutually interacted, severely lowering the patient’s life quality. Acupuncture intervention is effective in relieving psychological disorders such as depression and anxiety. But, if it works in reducing the negative affection of pain, current evidence is quite not enough.The Amygdaloid nucleus is one of the central "pain neuromatrix" serving sensory and negative affective components of pain. It plays a critical role in pain-induced aversion, and promotes the generation and maintenance of chronic pain. The amygdala contains a variety of biologically active molecules involving the regulation of pain, for example, corticotropin releasing factor (CRF) receptor protein, glutamate, opiod peptides, norepinephrine, etc. The CRF is a family of related neuropeptides and has a variety of physiological effects on stress and anxiety, vasoregulation, thermoregulation, growth and metabolism, etc. The glutamate, one of the excitatory neurotransmitters in the central nervous system, distributes widely in the central nervous system, participating in the transmission of nociceptive signal transmission, and generation and development of pain. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in adult mammalian central nervous system (CNS), and its receptors are the targets of analgesic drugs in clinical practice. All these molecules participate in the pain processing in the CNS.Our previous studies showed that the cholinergic system in both hippocampus and hypothalamus were involved in the process of acupuncture analgesia. The amygdaloid nucleus and hippocampus belong to the limbic system of the brain, serving emotion, memory and other physiological activities in the human body. The present study is aimed at exploring the function of Amygdaloid nucleus in mediating analgesic effect and negative affection-relief effect of electroacupuncture (EA) stimulation of Zusanli (ST36)-Yanglingquan (GB34) by regulating the expression of CRF receptor (CRF-1R, CRF-2R), glutamate NMD A receptors (NR2A,NR2B), and GABA receptors (GABAAR, GABACR) in the Amygdala in CCI+negative affection model rats, so as to reveal the possible targets of EA analgesia under chronic neuropathic pain conditions.Materials and Methods1) Part1Experiments were separately performed in 36 male Wistar rats which were randomized into normal control, CCI model and EA+CCI; normal control, negative affection (NA) model and EA+NA groups (n=6 in each group). The neuropathic pain model was established by ligature of the left sciatic nerve (to induce chronic compressive injury, CCI), and the NA model established by ligature of the left sciatic nerve combined with repeated skin stimulation (acupuncture needle pricking+direct current stimulation) of the paw, once daily for 3 days. EA (2Hz/15Hz,1 mA) was applied to bilateral ST36-GB34 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 3760. At the end of each experiment, the tissues of the right Amygdaloid nucleus were sampled for detecting the expression levels of CRF-1R, CRF-2R, NMDA-NR2A, NR2B, GABAA and GABAc genes using quantitative RT-PCR.2) Part 2Thirty-two male Wistar rats were randomized into four groups:normal control, CCI+NA (negative affection) model, CCI+NA+EA, and CCI+NA+AEA (anesthesia+EA, n= 8 in each group). The neuropathic pain (CCI) model was established in the same way to those mentioned above. The conditioned place aversion (CPA) model was established by CCI plus repeated skin electrical stimulation of the footplate in a conditioning test apparatus, once daily for 3 days. EA (2Hz/15Hz,1mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 7 days. Rats of the AEA group were given with EA under halothane anesthesia. The expression levels of CRF-1R, CRF-2R, NMDA-NR2B, mGluR7, GABAA and GABAB receptor proteins in the Amygdala were determined using Western blot.3) Statistics and analysis of data:All data were presented as mean± standard deviation (M±SD), and analyzed by one way ANOVA, followed by Least significant difference test for comparing data between every two groups. P<0.05 was considered statistically significant.Results:1) Part 1In comparison with their individual normal control group, PWL difference (PWLD) values of the bilateral paws of CCI model and CCI+NA model groups were significantly increased (P<0.05), suggesting a decrease of the thermal pain threshold. Following EA stimulation of ST36-GB34 for 7 days, the thermal pain thresholds were significantly higher in both EA+CCI and EA+NA than in the CCI and CCI+NA groups respectively (P<0.05), suggesting an apparent pain relief.RT-PCR results indicated that compared to the control group, the expression levels of CRF-1R, CRF-2R, NR2A and NR2B genes were apparently increased in the CCI model group (P<0.05), and those of CRF-1R, CRF-2R, NR2A, NR2B, GABAa and GABAc genes were remarkably down-regulated in the NA model group (P<0.05). After EA intervention for 7 days, the expression levels of CRF-2R, NR2A and NR2B genes were significantly down-regulated in the EA+CCI group, while those of CRF-2R, NR2A, NR2B, GABAa and GABAc genes were obviously up-regulated in the EA+NA group (P<0.05).2) Part 2In comparison with the normal control group, after CPA model establishment, PWL difference (PWLD) values of CCI+NA model group were significantly increased (P<0.001). Following EA intervention for 5 and 7days, PWLD values of both CCI+NA+EA and CCI+NA+AEA groups were considerably decreased in comparison with the CCI+NA model group(P<0.05), suggesting a pain relief.In comparison with the normal control group, the time spent in the CPA-paired compartment of CCI+NA group was significantly shorter (P<0.001), suggesting a place aversion to the CCI-paired compartment. Following EA for 5 and 7days,the time spent in the CPA-paired compartment of both CCI+NA+EA and CCI+NA+AEA groups were considerably increased, especially the CCI+NA+EA group in comparison with the CCI+NA group (P<0.001), demonstrating a relief of negative affection. Western blot assay indicated that compared to the control group, the expression levels of CRF-1R, CRF-2R, NR2B, mGluR7, GABAA and GABAB receptor proteins have no remarkable changes in the CCI model, CCI+NA+EA and CCI+NA+AEA groups (P>0.05)Summary1. Repeated EA stimulation of ST36-GB34 is effective in reducing neuropathic pain and negative affection in CCI and CCI+negative affection rats.2. EA stimulation of ST36-GB34 can down-regulate the expression levels of CRF-2R, NR2A and NR2B genes, and up-regulate the expression of CRF-2R, NR2A, NR2B, GABAa and GABAc genes in the amygdale in neuropathic pain and CCI+NA rats.3. EA stimulation of ST36-GB34 has no significant effect on expression levels of CRF-1R, CRF-2R, NR2B, mGluR7, GABAA and GABAB receptor proteins in pain-induced aversion rats, but this conclusion remains to be confirmed future. |
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