Research reportSpinal P2X7 receptor mediates microglia activation-induced neuropathic pain in the sciatic nerve injury rat model
Highlights
► BBG attenuated both mechanical allodynia and thermal hypersensitivity induced by CCI. ► P2X7R expression was increased in dorsal horn of spinal cord of CCI rats. ► Spinal P2X7R expression was highly restricted to microglia. ► Microglia activation was inhibited by blocking P2X7 receptor in CCI rats.
Introduction
Neuropathic pain is a common and severely disabling state, affecting millions of people worldwide [1]. It typically develops when peripheral nerves are damaged due to surgery, bone compression in cancer, diabetes or infection [2], [3]. Following nerve injury, there are many molecular and cellular changes of the peripheral and central nervous systems, among which ATP and its purine receptors have been widely studied.
It is well-known that adenosine 5′-triphosphate (ATP) can serve as an important chemical gliotransmitter that mediates a broad range of physiological and pathological processes in the nervous system [4]. Accumulated evidence indicates that ATP and its purine receptors are involved in the regulation of neuropathic pain [5], [6]. P2 purinoceptors were divided into two different categories: the ATP-gated ionotropic P2X family and the G protein-coupled (metabotropic) P2Y receptors [7]. These two types of P2 purinoceptors are widely distributed in the sensory nervous system and exhibit various effects both at neuronal and glial cells [8], [9], [10]. In the spinal cord, much emphasis has been focused on the potential role of P2X receptors in sensory processing in the dorsal horn, because the sensory neurons for pain, touch, and temperature are located in this area. However, the exact role of these receptors in the neuropathic pain remains unclear.
The P2X7R is a unique member of the P2X receptor family, because it is activated only by high concentrations of ATP (>100 μM) and its prolonged exposure to ATP has been shown to form a much larger pore than any other P2X channel [11]. P2X7R is primarily expressed in microglia and peripheral macrophages, a recent study has demonstrated that P2X7R is required for the activation and proliferation of microglia, suggesting that it regulates immune function and inflammatory responses [12]. Studies have shown that activation of the P2X7R can promote the release of some cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and some superoxide products, all of which play important roles in the generation or maintenance of pain. Chessell et al. found that hypersensitivity is completely absent to both mechanical and thermal stimuli in P2X7 gene-ablated (−/−) mice, whilst normal nociceptive processing is preserved [13]. Development of new potent and selective P2X7R antagonists also indicated a role for P2X7Rs in the onset and persistence of certain types of chronic pain [14], [15], [16]. Although these studies have revealed a role for P2X7Rs in the development of neuropathic pain, where alteration of P2X7R pathway is involved in the pathology of neuropathic pain is unclear.
A recent study by Chu et al. showed that in the dorsal horn of spinal cord, microglial P2X7 receptors signaling pathway was involved in the induction of long-term potentiation in response to spinal nociceptive stimuli [17]; however, how about the role that microglial P2X7R plays in the chronic constriction injury (CCI) of the sciatic nerve remains to be elucidated. In order to address this question, in this study we observed the expression of P2X7R in spinal cord dorsal horn in neuropathic pain induced by CCI, and explored the relationship of expression of P2X7R with tactile allodynia and thermal hypersensitivity after peripheral nerve injury. The selective P2X7R antagonist, Brilliant Blue G (BBG), was used to indicate that activation of P2X7Rs in spinal microglia participates in the pathogenesis of CCI-induced neuropathic pain.
Section snippets
Materials and methods
All experiments were performed in strict accordance with the ethical guidelines of the International Association for the Study of Pain [18], and also approved by the Ethical Committee for Animal Research of Third Military Medical University.
CCI-induced mechanical allodynia was attenuated by the selective P2X7R antagonist BBG
CCI model is a well-established and widely-used preclinical model of neuropathic pain. In this study we revealed profound differences mechanical sensitivity between groups after nerve ligation (Fig. 1). Mechanical allodynia was recorded as a significant reduction of the withdrawal threshold in ipsilesional hindpaw of rats after ligation when compared to that of the naïve controls and the sham-operation group (P < 0.001). Naïve controls and the sham-CCI group were relatively stable and had no
Discussion
In the present study, the CCI model was used to investigate neuropathic pain, and we observed expression of P2X7R in dorsal horn of L4–L6 spinal cord after nerve injury. Our data from Western blot and immunohistochemistry showed that P2X7R expression was increased in the ipsilateral spinal cord after nerve injury, 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
Conclusions
Our results indicate thatP2X7R expression was increased in the ipsilateral spinal cord after sciatic nerve ligation and was highly restricted to microglia, and CCI-induced mechanical allodynia and thermal hypersensitivity was reversed by intrathecal administration of a selective P2X7 antagonist BBG. In addition, up-regulation of OX42 expression was also observed in the ipsilateral spinal cord of CCI rats, but the number of reactive microglia was decreased and distribution of microglia was less
Competing interests
Authors declare that they have no competing interests.
Acknowledgments
This work is supported by the National Natural Science Foundation of China (No. 30970974). We are very grateful to the Ms. Wei Sun and Li-Ting Wang (Central Laboratory, Third Military Medical University) for their technique assistance in laser scanning confocal microscopy.
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