Preliminary Study Of The Function And Mechanism Of Microglial P2X₇ Receptor In Neuropathic Pain

Neuropathic pain is a common and severely disabling disease, affecting millions of people worldwide. The dorsal horn of spinal cord plays an important role in nociceptive information transmission from the periphery to central nervous system, because the sensory neurons for pain, touch, and temperature are located and many neuroactive substances and pain-ralated receptors are distributed in this area. Therefore, the expression of pain-related molecules in the dorsal horn after nociceptive stimulation should be observerd to increase our understanding of the development and maintenance of neuropathic pain. Accumulated evidence indicates that spinal microglia activation is contributed to neuropathic pain, but related cell and molecular mechanism of microglia activation in neuropathic pain remain unclear.Adenosine triphosphate (ATP) is an important neurotransmitter on nociceptive transmission in spinal cord. ATP play important role in CNS by binding with and acting on P2 purinocetor, which includes 7 subtypes of ATP-gated ionotropic P2X receptor and the 13 subtypes of G protein-coupled P2Y receptor. The P2X₇ receptor (P2X₇R) is a unique member of the P2X receptor family, which was indicated that involved in the transmission of nociceptive information in the spinal cord. The P2X₇R are widely distributed in the sensory nervous system and exhibit various effects both at neuronal and glial cells. But P2X₇R expression in spinal cord is to debate. ATP are released or leaked from injured-tissue cells as well as neurons after nerve injury in neuropathic pain. It is discovered that microglia activated by ATP can release a variety of proinflammatory cytokines and neuroactive substances. Relatively little insight has been gained concerning the relation between P2X₇R and spinal microglia activation,and the following mechanism of microglia activated by P2X₇R in neuropathic pain need to be further study.This study will use chronic constriction of the sciatic nerve (CCI) rat model, combine in vivo and in vitro experiment and apply some methods include morphology, molecular biology and [Ca²⁺]i image , to observe the intracellular concentration of Ca²⁺ and release of amino acid in microglia after P2X₇R activated and investigate the relationship between P2X₇ receptor and the activation of cultured microglia ; to observe the expression of P2X₇R in spinal cord dorsal horn in neuropathic pain induced by CCI, explore the relationship of expression of P2X₇R with spinal microglia activation and with tactile allodynia and thermal hypersensitivity after peripheral nerve injury, and speculate the effects of P2X₇R-mediated microglia activation in CCI-induced neuropathic pain. This study will lead to a better understanding of the molecular mechanisms of neuropathic pain and provide clues for the development of new therapeutic drugs.Methods:The expression of CD11b, P2X₇R and SR in cultured BV-2 microglia line cell were observed by using immunofluorescence. The changes of CD11b and SR expression after ATP and BzATP administration in microglia were observed by using immunohistochemical staining and analyzing by Image Prop–plus 6.0. Changes of [Ca²⁺]i after ATP and BzATP administration in microglia were detected with CLSM using fluo-3/AM as a calcium fluorescent indicator. ATP and BzATP -evoked amino acid release were detected by using HPLC. The changes expression of P2X₇R and OX42 in cultured BV-2 microglia line cell were observed by using immunohistochemical staining and western blot. Double-labeled immunofluoresence were used to determine the cell type that spinal P2X₇R expressed in. Mechanical allodynia was assessed using von Frey hairs (vFh) and thermal sensitivity measurement was carried out using Hargreaves methods in all groups of rats.Results:Part one:1.Cultured BV-2 microglia cell line was labeled by CD11b, and ratio of positive cell is more than 99 percent, which indicated that the cell line was suit to use in the following experiments. There is direct immunofluoresence evidence that the P2X₇R protein is expressed in cultured BV-2 microglia cell line, and the positive was located in cell body;2.ATP (0.1μmol/L, 1μmol/L) did not evoke an increase of [Ca²⁺]i in cultured BV-2 microglia cell line, but ATP (>10μmol/L) can increase the [Ca²⁺]i of cultured BV-2 microglia cell line; BzATP can evoked a long-lasting increase in the [Ca²⁺]i of cultured BV-2 microglia cell line even at 1μmol/L, a concentration at which ATP did not evoke the increase。3.Both of ATP and BzATP (100μmol) can evoked an increase in [Ca²⁺]i of microglia, which was blocked by 20 min pretreatment with an selective P2X₇ antagonist BBG(100nmol/L).4.ATP and BzATP (100μmol) was shown to evoke an increase in [Ca²⁺]i in the presence of extracellular Ca²⁺, but the increase was not observed in the absence of extracellular Ca²⁺.5.There is direct immunohistochemical evidence that ATP, BzATP -induced the increases of CD11b expression in cultured BV-2 microglia cell line, and 20 min pretreatment with BBG can inhibit the increase of CD11b expression.Part two:1.ATP and BzATP (100μmol) ATP and BzATP increase the release of glycine, after 10min treatment in microglia, which can inhibit by 20 min pretreatment with BBG; but ATP and BzATP (100μmol) have no effect on the release of aspartic acid and glutamate;2.BzATP–evoked glycine release in microglia was observed both in the presence of extracellular Ca²⁺ and the absence of extracellular Ca²⁺. After 20 min pretreatment with a selective protein kinase C antagonist Che (100μmol), BzATP–evoked glycine release was partly inhibited, but the concentration of extracellular Glycine still more than control. But BzATP–evoked glycine release was not changed by 20 min pretreatment with a selective protein kinase A antagonist H-89 (100μmol);3.ATP and BzATP (100μmol) have no effect on the release of D-Serine in microglia at 10min, but can increase the release of D-Serine in microglia after 24h, which indicated that ATP and BzATP evoked D-Serine release is due to synthesis of D-Serine. ATP and BzATP evoked D-Serine release in microglia can be blocked by 20 min pretreatment with BBG;4.BzATP–evoked D-Serine release in microglia was observed both in the presence of extracellular Ca²⁺ , but the increase was partly inhibited in the absence of extracellular Ca²⁺. After 20 min pretreatment with che(100μmol), BzATP–evoked D-Serine glycine release was partly inhibited, but the concentration of extracellular D-Serine still more than control. But BzATP–evoked D-Serine release was not changed by 20 min pretreatment with H-89(100μmol);5.There is direct immunohistochemical evidence that SR protein is expressed in cultured BV-2 microglia cell line, and co-expression of P2X₇R was observed in microglia; ATP, BzATP -induced an increases of SR expression in cultured BV-2 microglia cell line,which can inhibit by 20 min pretreatment with BBG.Part three:1.Mechanical allodynia and thermal hypersensitivity developed within 3 days (d), peaked at 14 d and persisted for 21 d, and showed a significant different when compared to that of the na?ve controls and the sham-operation group. Na?ve controls and the sham-CCI group were relatively stable and had no significant difference in the withdrawal threshold during the whole time course examined.2.The expression of P2X₇R protein in the ipsilateral spinal cord was markedly increased after nerve injury. A significant increase was observed from day 7 after injury; P2X₇R protein levels peaked on day 14. The most significant difference was observed at I-IV layers of the dorsal horn, which is one of the major areas for nociceptive signaling processing, and the time course of the change in P2X₇R levels in the dorsal horn of spinal cord and the difference in P2X₇R levels matched the emergence of mechanical allodynia and thermal hypersensitivity.3.The results of double-labeled immunofluorescence showed that P2X₇R immunofluor- escence were not double-labeled with MAP2 or GFAP (Fig. 5). Instead, almost all P2X₇R-positive cells were double-labeled with OX42, which indicated that the P2X₇R expression is highly restricted to microglia in the dorsal horn of spinal cord after nerve injury.4.In vehicle (saline)-treated rats, the PWMT and PWTL was decreased after CCI, which were not significantly different compared to untreated CCI rats. In contrast, repeated intrathecal administration of BBG markedly suppressed this decrease in PWMT and PWTL after nerve injury, which has shown significance after nerve ligation from 3 d to 14 d compared to saline-treated rats and untreated CCI rats. But intrathecal administration of BBG could not completely inhibit thermal sensitivity and mechanical allodynia in neuropathic pain, which has also shown significant differences compared to naive control.5.Immunohistochemical results have shown that OX42 protein expression up-regulates post-surgery, and was significantly higher in the ipsilesional dorsal horn of spinal cord after nerve injury. Positive cells exhibited reactive microglia morphology and surround the dorsal horn following nerve ligation. OX42 expression was decreased significantly compare with saline treated rats, when we blocked P2X₇R activation in L4–6 spinal cord using the selective P2X₇R antagonist BBG. The data also showed that in BBG treated 14d rats, distribution of microglia was not as intensive as in saline treated 14d rats or CCI rat, and less microglia exhibited hypertrophic morphology, although still more than in na?ve controls.Conclusions:1.P2X₇R protein is expressed in cultured BV-2 microglia, and participate in the ATP and BzATP-induced microglia activation.2.P2X₇R receptor mediate the [Ca²⁺]i responses induced by ATP and BzATP, which may attributed to the effect of increasing Ca ²⁺ influx.3.ATP and BzATP can increase Gly release from microglia, which are mediated via P2X₇R, but has no effect on Asp and Glu release from microglia. BzATP–evoked Gly release was Ca²⁺-independent manner and partly mediated by PKC, but PKA are not related to BzATP–evoked glycine release;4.ATP, BzATP activated P2X₇R in a long time can increase the release of D-Serine in microglia. BzATP–evoked the release of D-Serine was partly inhibited by removal of extracellular calcium and mediated by PKC, but PKA are not related to BzATP–evoked D-Ser release;5.SR protein is expressed in cultured BV-2 microglia, and ATP, BzATP increase the release of D-Serine in microglia,which may attributed to the increased expression of SR induced by activating P2X₇;6.P2X₇ expression was highly restricted to microglia, and significant increased in CCI rats model. P2X₇R mediated microglia activation induced by CCI, and participated the development of mechanical allodynia and thermal hyperalgesia in CCI-induced neuropathic pain.In summary, these results indicated that spinal P2X₇ receptor was expressed on microglia and participated in development of neuropathic pain. It was also indicated that ATP activated microglia and trigged release of Gly and D-Ser via P2X₇ receptor, which may be the molecule mechanism of P2X₇ mediated microglia activation to regulation neuropathic pain. Thus, blocking of microglial P2X₇R in spinal cord might represent a therapeutic strategy for treating neuropathic pain.