| Neuropathic pain results from direct damage and dysfunction of the peripheral or central nervous system. Neuropathic pain characterized by spontaneous pain, hyperalgesia, allodynia and paresthesia is a disturbing disease which incidence is over1%. Besides the suffering in the injuried side, previous studies reported that spontaneous pain and hyperalgesia also appeared in the contralateral side. This phenomenon is named as bilateral hyperalgesia.In the recent years, to understand the mechanisms underlying bilateral hyperalgesia has become a hot topic. Many pathways may be involved in the generation and maintenance of bilateral hyperalgesia. The pivotal role of glia-centered neuroinflammation in the development and maintenance of bilateral hyperalgesia has been recognized gradually. Astrocytes, which consist of the greatest part of glia in the central nervous system, have been found under activated state for long term (>5months) in the spinal cord of rats with neuropathic pain. Furthermore fluorocitrate, an inhibitor of astrocytes, administered intrathecally could attenuate neuropathic pain, while minocycline, the inhibitor of microglia, has little effect on the NP. Thus it has been widely accepted that the activated astrocytes play a pivotal role in the development of neuropathic pain. Nonetheless, the exact effects exerted by astrocytes in the neuroptahic pain are still elusive until now. Some scholars have proposed that gap junctions existed widely between astrocytes might involved in the activation of astrocytes under neuropathic pain. So further exploring the role of gap junctions on the activation of astrocytes will help to understand the mechanisms underlying the development of neuropathic pain.Gap junctions are specialized intercellular connections between the membranes of adjacent cells, which are the only direct material and information exchange channel between cells. The role of them is to bufferring ion concentration, to forming electrical synapses and to participating in signal transmission. Through the wide gap junctions, a network was formed by astrocytes. In peripheral and central nervous system, few neurons and virtually all astrocyts can express connexin, and the gap junctions between astrocytes are far greater than neurons. Microglia can not express connexion. When astrocytes are activated by nociceptive signals, channels at gap junction are open, the concentration of Ca2+in astrocytes is rapidly increased. Nociceptive signals are transmitted to the neighboring astrocytes, which can make the neighboring astrocytes activate. This phenomenon is named as oscillation or calcium waves. Calcium waves can make the astrocytes release ATP which can combine with the ATP receptors on neurons. After that the neurons were activated and resulted in the maintenance of pain. Furthermore, it has been proposed that under chronic pain state noxious stimulus signal could induce the form of Ca2+waves in astrocytes, then diffuse in the astorcytes network through GJ, subsequently activate astrocytes widely and release a great amount of products, consequently result in the development of bilateral hyperalgesia. Until now this proposal is still not proved by enough evidence.As for the products released by activated astrocytes, previous studies have indicated as:chemokines, prostaglandins and inflammatory cytokines and so on. Among inflammatory cytokines, TNF-a and IL-1β have been suggested to bind the receptors directly or indirectly on the surface of neurons or other immune cells, then regulate the synthesis and release of other inflammatory cytokines, finally the generation of neuropathic pain. Dong-ho youn found that intrathecal injection of TNF-a can induced hyperalgesia in rats. Pinhui Miao also observed that the levels of TNF-a in PNI were continuing increased and were mainly distributed in the astrocytes, Periphereal or intrathecal injection of TNF-a antibody can relate bilateral hyperalgesia, which shows that TNF-a plays an important role in the bilateral hyperalgesia. In xenopus, the sheath of the sciatic nerve, spinal cord and brain by injection of IL-1β can lead to the emergence of animal hyperalgesia, and peripheral or intrathecal injection of IL-1β antibody can alleviate animal hyperalgesia, which indicate that IL-1β plays an important role in the generation of hyperalgesia. The role of inflammatory cytokines in pain may be related to the promotion of the pain fibers to release calcitonin gene-related peptide, substance P and exciting nociceptive neurons, which can increase the release of pain neurotransmitters by primary afferent nerve and increase the excitability of pain transmission neurons to extend the pain transmission.Many animal models have been established to study neuropathic pain. Some models such as:sciatic nerve inflammation (SNI) model, chronic sciatic nerve constriction injury (CCI) model, and L5spinal nerve transection (SNT) model and so on could express bilateral hyperalgesia. L5spinal nerve transection model is a frequently used one by our lab, in which obvious hyperalgesia is observed in the injuried side1day after nerve transection and maintained for4monthes, and less hypergesia is observed10days after transection in the contralateral side. This phenomenon quite resembles the contralateral symptoms in patients with neuropathic pain.In this experiment, using the rat L5spinal nerve transection model we observed the effect of intrathecal injection of CBX, a widely used GJ inhibitor, on bilateral mechanical hyperalgesia and observe the expression of GFAP, TNF-a and IL-1(3in the lumbar spinal dorsal horn to further explore the possible role of GJ, astrocytes and cytokines on the development of bilateral mechanical hyperalgesia.OBJECTIVETo observe the effects of intrathecal injection of carbenoxolone (CBX) on bilateral hyperalgesia and expression of astrocytes surface marker-GFAP (glial fibrillary acidic protein) and pro inflammatory cytokines as:TNF-α and IL-1β in bilateral spinal horns in rats with L5spinal nerve transection (SNT). To explore the possible roles of GJ、astrocytes and cytokines in the generation of bilateral hyperalgesia under neuropathic pain.METHODSSixty male Sprague-Dawley rats were randomly divided into five groups(n=12): group I received sham surgery then treated with saline; group II recieved SNT then treated with saline; groupIII received SNT then treated with0.05p.g CBX; group IV received SNT then treated with0.5μg CBX group Ⅴrecievd SNT then treated with5μg CBX. Group I underwent a sham operation. Group II to V maked the model of SNT according to Kim and Chung. Rats were anesthetized by2%pentobarbital. Routine disinfection, prone to tied up, belly pad to pad, full abdominal song, along the L4-6spinal midline skin incision, blunt dissection the left side of the spinal muscle tissue, cut into transverse process, expose the L5spinal nerve using3-0silk ligature and cut off the left side of L5spinal nerve. Treatment was undertaken with10μl volume as a single intrathecal injection on postoperative day10. Mechanical withdrawl thresholds were measured1d before operation,1d,3d,5d,7d and10d after sugery,1h before intrathecal administration, and1h,2h,4h,6h after intrathecal administration. Lumbar spinal cord was obtained2h after intrathecal administration to determine the expressions of GFAP by immunohistology、TNF-a and IL-1β by ELISA in bilateral spinal dorsal horns.The data was analyzed by SPSS13.0statistic software.Values were presented as means±SD.Comparisons among groups were performed by one way ANOVA analysis. If the variance to be equalized, two sets data were analyzed with LSD test. If the variance to be not equalized, two sets data were analyzed with Dunnett’s T3test. Repeated measurement datas were performed Repeated Measures. If the datas meet the assumption, the datas were analyed with Sphericity Assumed. If the datas don not meet the assumption, the datas were analyed Greenhouse-Geisser. Comparisons among groups were performed by one way ANOVA analysis. Comparisons among different times were performed Repeated Measures. P≤0.05was considered significant.RESULTS The results of the bilateral MWT Before surgery, there was no significant difference of the MWT among the five groups(P>0.05);Compared with the MWT before sugery, the value on postoperative days in group I was no marked difference (P>0.05), Rats in group Ⅱ-Ⅴ developed allodynia in the operated paw, which appeared on postoperative day1and was significantly decreased at5to10days (P<0.05), while the contralateral MWT was significantly decreased at10d (P<0.05); Compared with the MWT1h before intrathecal administration on day10, the values at1h,2h,4h,6h after administration of group Ⅱand Ⅲ were no marked difference (P>0.05). The ipsilateral MWT in group Ⅳ was no significant difference at1h,2h,4h after administration (P>0.05) while the contralateral MWT was began to increase at1h after administration (P<0.05), at2h the MWT was significantly increased (P<0.05), at4h the MWT began to decline but was still higher than before injection (P<0.05).There was no significantly difference at6h (P>0.05). In group V the bilateral MWT was significantly improved at1h,2h,4h after administration (P<0.05). There was no significantly difference at6h after administration (P>0.05)The results of the bilateral GFAP The results of immunohistochemistry showed that GFAP staining in the bilateral spinal cord dorsal horn in group Ⅱ and Ⅲwas significantly increased compared with the group. Compared with group I, GFAP staining in the ipsilateral spinal cord dorsal horn in group IV was significantly enhanced. Compared with group II, GFAP staining in the ipsilateral spinal cord dorsal horn in group IV was no significantly difference. Compared with group II, GFAP staining in the contralateral spinal cord dorsal horn in group IV was significantly decreased. Compared with group II, GFAP staining in the bilateral spinal cord dorsal horn was significantly decreased in group V. Compared with the group I, the positive cells of GFAP in the bilateral spinal cord dorsal horn in group II and III were significantly increased (P<0.05). Compared with group I, the positive cells of GFAP in the ipsilateral spinal cord dorsal horn in group IV were significantly increased (P<0.05). Compared with groupⅡ, the positive cells of GFAP in the ipsilateral spinal cord dorsal horn in group Ⅳ were no significantly difference (P>0.05), while the positive cells of GFAP in the contralateral spinal cord dorsal horn in group Ⅳ were significantly decreased (P<0.05). Compared with group II, the positive cells of GFAP in the bilateral spinal cord dorsal horn were significantly decreased in group V (P<0.05).The results of the bilateral TNF-a and IL-1β The results of ELISA showed that the levels of TNF-a and IL-1β in group II and III group were significantly increased comparing with group I (P<0.05). Compared with group II, the levels of TNF-a and IL-1β in the ipsilateral spinal cord dorsal horn in Ⅳ group were no significantly difference (P>0.05), while the levels in the contralateral spinal cord dorsal horn were significantly decreased (P<0.05). Compared with group Ⅱ, the levels in the bilateral spinal cord dorsal horn of group V were significantly decreased (P<0.05).CONCLUSIONS1. L5spinal nerve transection resulted in bilateral mechanical hyperalgesia, which appeared in the operated paw on postoperative day1and reached apparently on day5to10, while in the contralateral side appeared on postoperative day10.2CBX could dose-dependently improve the bilateral MWT with the significantly decrease of the levels of GFAP、TNF-α and IL-1β in the spinal doral horn. These suggested that GJ、astrocytes and cytokines may be involved in the generation of bilateral hyperalgesia under neuropathic pain... |
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