| Neuropathic pain has been redefined as "pain arising as a direct consequence of a lesion or disease affecting the somatosensory system". It was induced by various peripheral or central nervous system diseases, such as trigeminal neuralgia, postherpetic neuralgia, painful diabetic neuropathy, Parkinson’s disease and so on. In china, about 0.9 hundred million people have been suffered from neuropathic pain, according to the incidence of 8% in people. Moreover, increasing evidence indicated that chronic neuropathic pain, as a long-term stressor could induce depression, anxiety and other mental disorders. It causes huge suffering and mental impairments for the patients, and now has become an important public health problem. So, it is significant to further explore the pathogenesis and therapy for neuropathic pain and pain-induced emotional disorders.As we know, neuroinflammation plays a critical role in the processing of neuropathic pain. When the peripheral nerves suffering the noxious stimulus, sustained afferent nociceptive information could induce neuroinflammation in spinal cord and facilitated the central sensitization. Then the excitability of pain transmission neurons was increased and the pain signals can be further amplified and maintained. The neuroinflammation in spinal cord was characterized by the activation of glial cells and the production of excessive inflammatory cytokines (IL-1β, TNFα, IL-6, etc.). IL-1β as an important pro-inflammatory cytokine plays a key role in the development of neuropathic pain behaviors. Studies have shown that in the spinal cord of rats with neuropathic pain, the transcription of IL-1β into its precursor molecules was increased. However, the inactive IL-1β precursor need to be cut into the mature IL-1β molecule by proteinase and then exerts its biological activity.The inflammasome-caspase-1 platform is one primary mechanism responsible for the maturation of IL-1β. Inflammasomes are groups of protein complexes that recognize diverse sets of inflammation-inducing stimuli such as pathogenic infection or tissue damage. When the inflammasome was activated, the protein component pro-caspase-1 was hydrolyzed into its mature caspase-1 form. Then the mature caspase-1 was released from the complex and cutting the pro-IL-1β molecule through its protease activity. Notably, in the rats with complex regional pain syndrome, the NALP1 inflammasome participated in the formation of peripheral sensitization. So, the effect of spinal NALP1 inflammasome in CCI neuropathic pain has been the first part of our research.Seeking effective treatments for the central neuroinflamination has always been the focus of our laboratory. Recent studies have found that lipoxin and its exogenous analogue aspirin-triggered carbon-15 epimer (ATL) exhibit powerful anti-inflammatory effects and act as endogenous "brake signals" in the inflammation reactions. Our previous study showed that ATL effectively inhibited the inflammatory cytokines, including IL-1β in the spinal cord of neuropathic pain rats. So, the effect of ATL on spinal NALP1 inflammasome in rats with neuropathic pain is one problem of our concern.Chronic pain, as a physical and psychological stressor, persistently disturbed the brain function. Patients with chronic pain frequently suffered from mental illnesses, particularly depression and anxiety. Clinically, the incidence of chronic pain induced-depression was up to 60%, so the mental damage produced by neuropathic pain cannot be ignored. Studies have shown that the neuroinflammation in brain, including glial activation and production of proinflammatory cytokines, was one important mechanism in neuropathic pain-induced mental disorders. Previous data also confirmed that IL-1β was significantly upregulated in the hippocampus of depressive rats. So, the question of the role of hippocampal NALP1 inflammasome in neuropathic pain-induced psychiatric disorders has been the second part of our research.Electroacupuncture (EA), as a traditional Chinese method, is widely accepted in neuropathic pain clinics and recent studies indicated that EA was increasingly applied to psychiatric diseases. Compared with the antidepressants, acupuncture showed a rapid onset and exhibited no toxic side effects, which provided a new opportunity for depression treatment. According to the previous results, EA displayed potent antidepressant-like effects in rats subjected to chronic unpredictable stimuli. However, whether EA has effects on the neuropathic pain induced depression and anxiety behaviors has been the third part of our research.According to the reported animal model of neuropathic pain, the presented study was aimed to:.(1) Effects of spinal NALP1 inflammasome on neuropathic pain during the first 7 days after surgery. (2) Effects of hippocampal NALP1 inflammasome on neuropathic pain-induced depression and anxiety-like behaviors. (3) Effects of Electroacupuncture (EA) on the depression and anxiety-like behaviors induced by neuropathic pain.The results are as follows:Part One. Effect of spinal NALP1 inflammasome on neuropathic pain1. Chronic constriction injury induced mechanical allodynia and thermal hyperalgesia in ratsSD male rats were divided into Normal group, Sham operation group and the model group subjected to neuropathic pain. Neuropathic pain was induced as the previous described. Briefly, the right common sciatic nerve was exposed by blunt dissection, and four ligatures (4-0 chromic catgut) were tied loosely around the sciatic nerve. "Sham surgery" referred to the procedure of exposing the nerve as above but without any nerve li gat ion. The paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were detected from day 2 to day 28 after CCI surgery. The results suggested that compared to the normal and sham group, the rats subjected to CCI showed a statistical decrease in the ipsilateral PWL and PWT.2. The activation and cellular localization of NALP1 inflammasome in the spinal cord of CCI rats.2.1 The activation of NALP1 inflammasome in the spinal cord of CCI rats.SD male rats were divided into Normal group, Sham operation group and model group. The ipsilateral L4-L5 segments of the spinal cord were quickly removed at day 1,3,7 after surgery. And the total protein was extracted to detect the mature IL-1β, mature caspase-1 and NALP1 inflammasome using the ELISA、Western blot and IP tests. In the western blot test, the mature IL-1β was significantly increased in the spinal cord of CCI rats at day 7 after surgery, and the mature caspase-1 was elevated at day 3. From day 3 to day 7, a significant aggregation of the protein components in NALP1 inflammasome was also detected in the IP test. However, there were no significant differences between normal and sham surgery rats.2.2 The cellular localization of NALP1 inflammasome in the spinal dorsal horn of CCI rats.The rats were divided into Normal group and Model group and the transverse sections of the L4-L5 spinal segments were cut in a freezing microtome at day 7 after surgery. The results showed that either in the normal or model group, the caspase-1 immunoreactivity within the spinal dorsal horn was co-localized with the neuron marker Neuron-specific Nuclear Protein (NeuN) and the astrocyte marker glial fibrillary acidic protein (GFAP) but not with the microglia marker CD11b. The levels of NALP1 immunoreactivity in the spinal cords of normal rats was low, and the CCI surgery upregulated NALP1 immunoreactivity in the NeuN-positive and GFAP-positive cells. Especially, the CCI surgery induced an increase in the immunoreactivity of caspase-1 and NALP1 within astrocyte at the superficial laminae of spinal dorsal horn.3. Effect of caspase-1 inhibitor on neuropathic pain in rats.To assess the involvement of inflammasome in the development of neuropathic pain, the caspase-1 inhibitor Ac-Tyr-Val-Ala-Asp-CMK (Ac-YVAD-CMK) was intrathecally administrated to CCI rats once a day from day 3 to day 6 after surgery. In the Hargreaves test, both 2 nmol and 20 nmol Ac-YVAD-CMK significantly attenuated the CCI-induced thermal hyperalgesia compared to the control DMSO treatment. In the ELISA test, the CCI rats that received 20 nmol Ac-YVAD-CMK also displayed a significant reduction in the level of spinal mature IL-1β compared to the rats received DMSO.4. Effect of ATL on spinal NALP1 inflammasome in CCI rats.ATL has exhibited its anti-inflammatory properties in many animal models. To detect whether ATL has an analgesic effect on CCI-induced neuropathic pain and to further reveal the underlying mechanism of its effect, we administered ATL to CCI rats at two different doses from day 3 to day 6 after the nerve injury.4.1 Effect of ATL on thermal hyperalgesia in CCI rats.SD male rats were divided into Normal group, Saline group, ATL (100 ng/day) group and ATL (200 ng/day) group. ATL (100 ng or 200 ng in 20μl) was administered daily from day 3 to day 6 after the CCI operation to evaluate its effects on the thermal hyperalgesia of CCI rats. In the Hargreaves test,200 ng ATL significantly reduced the CCI-induced thermal hyperalgesia compared to the control saline treatment, but 100 ng ATL showed no obvious pain-relieving effect on CCI rats.4.2 Effect of ATL on spinal NALP1 inflammasome activation in CCI rats.In the present study, we detected the effects of ATL on the mature IL-1β, mature caspase-1 and the combination of NALP1 inflammasome components via western blot, ELISA and IP tests. In the IP experiments, repeated ATL treatment reversed the increased aggregation of ASC, NALP1 and caspase-1 within the NALP1 inflammasome complex. In western blots, there was also a significant reduction in the level of mature caspase-1 and mature IL-1β after the CCI rats received 200 ng ATL.Summary:CCI initiated the activation of NALP1 inflammasome in spinal cord, leading to the cleavage of pro-caspase-1 and the maturation of IL-1β. The components of the NALP1 inflammasome complex were visible in the astrocytes and neurons of the spinal dorsal horn. Repeated intrathecal administration of ATL significantly relieved the CCI-induced thermal hyperalgesia and suppressed the spinal NALP1 inflammasome activation. Part Two. Effect of hippocampal NALP1 inflammasome on neuropathic pain-induced psychiatric disorders1. CCI induced depression and anxiety-like behaviors in ratsSD male rats were divided into Normal group, Sham operation group and Model group. To investigate whether the CCI rats developed depression-and anxiety-like behaviors, the FST and EPM tests were performed on day 7 and day 21, respectively. During the FST, CCI induced a marked. increase in the immobile time and an obvious reduction in the climbing time. In the EPM test, the percentage of open arm entries and the percentage of time spent in the open arms were all significantly decreased in the CCI group compared with the normal and sham surgery groups. Additionally, the open field test (OFT) was carried out to exclude the possibility that the lesion of the CCI surgery affects the performance of rats displayed in the FST and EPM tests. In the OFT, CCI rats displayed no reduction in the horizontal or vertical movements.2. The activation and cellular localization of NALP1 inflammasome in the hippocampus of CCI rats.2.1 The activation of NALP1 inflammasome in the hippocampus of CCI rats.SD male rats were divided into Normal group, Sham operation group and Model group. The bilateral hippocampi were quickly removed at day 3,7, 21 after surgery. And the total protein was extracted to detect the mature IL-1β, mature caspase-1 and NALP1 inflammasome using the ELISA、Western blot and IP tests. In the western blot test, the mature IL-1β was significantly increased in the contralateral hippocampus of CCI rats at day 7 after surgery, and the mature caspase-1 was also elevated at day 7. From day 7 to day 21, a significant aggregation of the protein components in NALP1 inflammasome was also detected in the hippocampus of CCI rats.2.2 The cellular localization of NALP1 inflammasome in the hippocampus of CCI rats.The rats were divided into Normal group and Model group and the brain were cut in a freezing microtome at day 21 after surgery. The results showed that either in the normal or model group, the caspase-1 immunoreactivity within the DG, CA1, CA3 area was primarily co-localized with the neuron marker NeuN and partly co-localized with the astrocyte marker GFAP but not with the microglia marker CD11b. Especially, after the rats subjected to the CCI surgery, the immunoreactivity of caspase-1 co-localized with NeuN was increased in each area.3. Effect of caspase-1 inhibitor on neuropathic pain-induced depression and anxiety like behaviors in rats.To assess the involvement of inflammasome in the development of neuropathic pain-induced emotional behaviors, the caspase-1 inhibitor Ac-YVAD-CMK was administrated into hippocampus once a day from day 7 to day 21 after the CCI operation. In the FST test,10 nmol Ac-YVAD-CMK significantly attenuated the CCI-induced depression-like behaviors compared to the control DMSO treatment. But there was no effect of 10 nmol Ac-YVAD-CMK on the anxiety-like behaviors in EPM. In the ELISA test,10 nmol Ac-YVAD-CMK also reduced the level of mature IL-1β in the hippocampus of CCI rats.Summary:The NALP1 inflammasome was activated in the hippocampus of CCI rats, leading to the cleavage of pro-caspase-1 and the maturation of IL-1β. The caspase-1 was visible in the astrocytes and neurons at the DG, CA1 and CA3 areas. Repeated intrahippocampal administration of YVAD to the CCI rats significantly relieved the CCI-induced depression-like behaviors and suppressed the IL-1β maturation in hippocampus.Part Three. Effect of repeated EA on neuropathic pain-induced psychiatric disorders1. Effect of EA on the depression and anxiety-like behaviors in CCI rats.To evaluate the curative effect of repeated EA on chronic pain-induced emotional behaviors, rats were divided into Normal group, Model group, Sham EA group and EA group. Additionally, the classic antidepressant fluoxetine was applied to another group of CCI rats beginning on day 7 after the surgery as comparison.In the FST, chronic EA treatment partially reversed the CCI-induced depression-like behaviors. EA at GV20-GB34 caused a significant reduction in the immobile time of CCI rats, but had no influence on the climbing time. Correspondingly, fluoxetine at the dose of 30 mg/kg for 14 consecutive days not only reduced the immobility time but also extended the climbing time of CCI rats.In the EPM test, EA treatment for continuous 2 weeks significantly improved the anxiety-like behaviors of CCI rats, characterized by an obvious increase in the percentage of open arm entries and the percentage of time spent in the open arms. However, chronic fluoxetine at either 10 mg/kg or 30 mg/kg produced no significant effect on the anxious behaviors of CCI rats.2. Effect of EA on the thermal hyperalgesia in CCI rats.The thermal hyperalgesia of rats receiving EA treatment was also monitored at day 3, day 7, day 14, and day 21 after the CCI surgery. Chronic EA at GV20-GB34 had no effect on CCI-induced thermal pain behaviors, which further illustrated that the therapeutic action of EA treatment on neuropathic-pain-induced emotional behavior was not due to an analgesic effect.Summary:After the CCI surgery, rats displayed significant depression-and anxiety-like behaviors. Repeated EA treatment completely abolished the CCI-induced anxiety-like behaviors and partially improved the depression-like behaviors in CCI rats.Conclusion:1. The NALP1 inflammasome was activated in the spinal cord of CCI rats. The components of the NALP1 inflammasome complex were visible in the astrocytes and neurons of the spinal dorsal horn. Repeated intrathecal administration of ATL to the CCI rats significantly relieved the CCI-induced thermal hyperalgesia and suppressed the spinal NALP1 inflammasome activation.2. The CCI rats displayed significant depression- and anxiety-like behaviors on 7 days after surgery. The NALP1 inflammasome was activated in hippocampus, leading to the cleavage of pro-caspase-1 and the maturation of IL-1β. Inhibition of the mature caspase-1 in hippocampus significantly relieved the CCI-induced depression-like behaviors and suppressed the IL-1β maturation.3. Repeated EA treatment effectively improved the CCI-induced depression and anxiety-like behaviors in rats. And in the present study, EA had an advantage over the fluoxetine in terms of the anxiolytic effect. |
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