| Neuropathic pain is defined as pain that caused by damage or disease that affects thesomatosensory system. It is a severe and common illness which has troubled people all overthe world.The spinal dorsal horn is the primary state in the central nervous system (CNS) fortransmission and modulation of pain-related information from the periphery to the CNS. Itis also the region of central axons of sensory neurons for pain, warm and touch and manyreceptors, neurotransmitters and neuroactive substances in this area. Hence, the research ondelivery and modulation of nociceptive information on the hierarchy of spinal cord willhave a great influence on the basis of occurrence and maintenance of neuropathic pain. Lotsof researches have proved that the activation of microglia in the spinal dorsal horn play animportant role in the occurrence and maintenance of neuropathic pain, however, the relativemolecular and cellular mechanism still unclear.Adenosine triphosphate (ATP) and its hydrolysis product, adenosine diphosphate(ADP), have been proved to be significant neurotransmitters on neuronal signaltransmission. ATP and ADP play important roles in CNS through the specific receptor, theP2purinergic receptors, which could be divided into P2X receptors and P2Y receptorsaccording to their physical characters. The7types of P2X receptors are ATP-gated inotropicchannels and the13types of P2Y receptors are G-protein-coupled receptors. Researchershave found the P2X7receptor (P2X7R) as a distinct member of the P2X receptor family andP2Y12receptor (P2Y12R) is a member of P2Y family, which are both expressed on thesurface of microglia and widely distributed in the CNS. There has been series of researchesindicated that both P2X7R and P2Y12R are participated in the delivery and modulation ofpain related information. When injuries occurred to the peripheral tissue, ATP will be released from the wounded cells or the sensory neurons and then can be hydralyated by theexocytase into ADP. Activated by ADP or ATP, a series of proinflammatory cytokines andneuroactive substances would be discharged from the activated microliga. There has beenlittle concentration on the relations between P2Y12R/P2X7R and activation of spinalmicroglia, as well as the following mechanisms, which need to be studied further.This research will use primary cultured microglia and the rat chronic constructioninjury(CCI) model, with experiments both in vivo and in vitro and combine methodscontaining morphology, ethology and calcium image, to detect the intracellularconcentration of Ca2+and release of proinflammatory cytokines from microglia after theactivation of P2Y12R or P2X7R; to investigate the relationship between P2Y12R/P2X7R andthe activation of primary cultured microglia; to observe the relationship of P2Y12R/P2X7Ractivation in dorsal horn of spinal cord in pain releated behaviors induced by administrationof P2Y12R/P2X7R agonist; to detect the effect of antagonist of P2Y12R/P2X7R on the CCIrats, which contains the expression of CD11b in the spinal dorsal horn and tactile allodyniaand heat hypersensitivity. According to this study, a more profound understanding ofintracellular mechanisms of neuropathic pain on the level of spinal cord will be gained andclues for the development of new treatment strategies will be got.Methods:By using immunofluorescence, the expression of P2Y12R, P2X7R and CD11b inprimary cultured brain cortex microglia was observed. The alteration of microglialmorphology after2MeSADP, AR-C66096+2MeSADP, BzATP or BBG+BzATPadministration into the culture medium of cultured microglia were observed by usingimmunohistochemical staining and the results was analyzed by the software, ImagePro–plus5.0. Changes of concentration of intracellular Ca2+after administrating2MeSADPinto the culture medium of microglia were observed with CLSM by using the calciumfluorescent indicator, fluo-4/AM. BzATP/2MeSADP-evoked IL-1β and TNF-α release weredetected by using ELISA. The immunofluorescence was used to detect the changes ofexpression of CD11b after2MeSADP, AR-C66096+2MeSADP, BzATP or BBG+BzATPadministrated intrathecally and while BBG/AR-C66096administrated intrathecally with ratsafter CCI surgery. Thermal hypersensitivity was executed with Hargreaves methods andmechanical pain threshold was measured by using von Frey hairs (vFh) in all groups of rats. Results:Chapter two:1. Primary cultured microglia cell expressed CD11b, and over90percent of cells waslabeled by CD11b, which manifested that we could use the cells we got was suitable in thenext experiments. The immunofluorescence results indicated directly that the primarycultured microglia cell expressed the P2Y12R protein, and the positive was located in cellbody.2. The agonist of P2Y12R,2MeSADP can evoked an increase in [Ca2+]iof microglia ina dose-dependent manner, which can be cut off by pretreatment with an selective P2Y12antagonist AR-C66096, which also is in a dose dependent manner.3. The surface and branches of the cultured microglia remarkably altered afteradministration of the P2Y12R agonist,2MeSADP, for24hours. And the function of2MeSADP can be reversed by the pre-treatment of AR-C66096.4. The concentration of interlukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) inthe supernatant increased after2MeSADP (100μM) administrated into the culture medium,which was blocked by30min pretreatment of AR-C66096.5.2MeSADP-evoked release of IL-1β can be blocked by the remove of extracellularCa2+. When administrated of G protein blocker, GDPβS, or the antagonist of phospholipaseC (PLC), neomycin, for30min, the release of IL-1β would be inhibited. While the processcould not be influenced by the protein kinase A antagonist, H-89.6. The concentration of TNF-α remarkably decreased without extracellular calcium inthe culture medium.2MeSADP-evoked release of TNF-α can be blocked by a series ofintracellular signal blocker, which includes G protein blocker, GDPβS, PLC antagonist,neomycin, PKA antagonist, H-89and p38MAPK blocker, SB203580. However, the proteinkinase C antagonist,Che, has no influence on the process.Chapter three:1. The immunofluorescence results showed that the P2X7receptor mainly expressed onthe membrane and cytoplasm of primary cultured microglia.2. The shape of the cultured microglia altered obviously after administration of theP2X7R agonist, BzATP, for24hours. And the function of BzATP can be reversed by thepretreatment of BBG. 3. BzATP increases the release of IL-1β and TNF-α, after1hour treatment withmicroglia, which can be inhibited by30min pretreatment with BBG.4. BzATP-evoked release of IL-1β was cut off by the clearance of extracellular Ca2+from the cultured medium. The effect of BzATP would altered by the pretreatment of PKAantagonist, H-89and GDPβS, neomycin didn’t show any influence.5. The concentration of TNF-α decreased without Ca2+in the cultured medium eventhough the cultured microglia was exposed to BzATP. BzATP-evoke release of TNF-α wasblocked by30min pretreatment of PKA antagonist, H-89or PKC antagonist, Che. Theconcentration of TNF-α in the culture medium didn’t change when GDPβS, neomycin orSB203580administrated into the culture medium.Chapter four:1. Mechanical and thermal pain related behaviors was appeared in3days(d), peaked at7d when administration of2MeSADP subarachnoidly and the pain related behaviors wouldchange into normal without any injection. The rats received injection ofAR-C66096+2MeSADP or AR-C66096alone had no obvious differences in the withdrawalthreshold during the whole time. And the rats received injection of AR-C66096after CCIsurgery could get less feeling of pain.2. The CD11b positive cells on the spinal dorsal horn on L4~6from the2MeSADPgroup was increased remarkably compared with the PBS group and peaked at day7. Thepositive results of CD11b in the CCI+AR-C66096group decreased than the control.3. Tactaile allodynia and thermal hypersensitivity progressed in3days(d), peaked at7dwhen administration of BzATP subarachnoidly and the pain related behaviors would changeinto normal without any injection. When injected BBG+BzATP or BBG alone, rats wererelatively stable and had no remarkable differences in the pain threshold in the whole period.The pain related behaviors of the rats, which got BBG administrated, would become lesssevere than the others.4. Expression of CD11b on the spinal dorsal horn on L4~6from the BzATP group wasincreased compared with the PBS group and peaked on day7. The level of CD11b woulddecrease in the CCI+BBG group. Conclusions:1. The primary cultured microglia expressed the P2Y12R protein, involved in theactivation of microglia induced by2MeSADP; increase of [Ca2+]ievoked by2MeSADPwas induced by the P2Y12R and may be related with the influx of extracellular calcium.2.2MeSADP can increase IL-1β and TNF-α release from microglia, which aremediated via P2Y12R. The release of IL-1β or TNF-α evoked by2MeSADP was in aCa2+-dependent manner and partly mediated by a series of other second messengermolecules.3. BzATP can increase the release of IL-1β and TNF-α from microglia, which aremediated via P2X7R. BzATP-evoked IL-1β or TNF-α release was Ca2+-independent manner,but PLC and G protein are not related to BzATP-evoked IL-1β release.4. The pain related behaviors was remarkably decreased and the expression of CD11bin the spinal dorsal horn increased after2MeSADP administrated intrathecally. And theadministration of P2Y12R antagonist, AR-C66096, may cause inhibition of the activation ofmicroglia and remission of pain behaviors caused by CCI surgery.5. The pain threshold was obviously decreased and the expression of CD11b in thespinal cord increased after P2X7R agonist injected intrathecally. And intrathecaladministration of P2X7R antagonist, BBG, could rival the decrease of mechanical andthermal pain threshold caused by the CCI surgery and inhibit the activation of microglia inthe spinal dorsal horn.In summary, our results investigated that spinal P2Y12receptor; P2X7receptor wasexpressed on microglia and participated in the activation of microglia under neuropathicpain. It was also indicated that P2Y12R or P2X7R triggered release of IL-1β and TNF-αfrom activated microglia may be the neuronal modulating basis to regulate the activity ofneurons when microglia was activated under neuropathic pain. Hence, intervention of thefunction of microglial P2Y12R/P2X7R in the level of spinal cord would be a possibletreatment for neuropathic pain in the future. |
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