Elsevier

Neuroscience

Volume 254, 19 December 2013, Pages 230-240
Neuroscience

Involvement of the spinal NALP1 inflammasome in neuropathic pain and aspirin-triggered-15-epi-lipoxin A4 induced analgesia

https://doi.org/10.1016/j.neuroscience.2013.09.028Get rights and content

Highlights

  • NALP1 inflammasome platform was activated in the spinal cord of CCI rats.

  • CCI-induced NALP1 inflammasome was located in spinal neurons and astrocytes.

  • ATL relieved the CCI-induced neuropathic pain behaviors.

  • ATL inhibited the CCI-induced activation of the NALP1 inflammasome.

Abstract

Neuroinflammation plays an important role in nerve-injury-induced neuropathic pain, but the explicit molecular mechanisms of neuroinflammation in neuropathic pain remain unclear. As one of the most critical inflammatory cytokines, interleukin-1β (IL-1β) has been regarded as broadly involved in the pathology of neuropathic pain. The inflammasome caspase-1 platform is one primary mechanism responsible for the maturation of IL-1β. Lipoxins, a type of endogenous anti-inflammatory lipid, have proved to be effective in relieving neuropathic pain behaviors. The present study was designed to examine whether the inflammasome caspase-1 IL-1β platform is involved in chronic constriction injury (CCI)-induced neuropathic pain and in lipoxin-induced analgesia. After rats were subjected to the CCI surgery, mature IL-1β was significantly increased in the ipsilateral spinal cord, and the inflammasome platform consisting of NALP1 (NAcht leucine-rich-repeat protein 1), caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) was also activated in spinal astrocytes and neurons, especially at the superficial laminae of the spinal dorsal horn; The aspirin-triggered-15-epi-lipoxin A4 (ATL), which shares the potent actions of the endogenous lipoxins, was administered to the CCI rats. Repeated intrathecal injection with ATL markedly attenuated the CCI-induced thermal hyperalgesia and significantly inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1β maturation. These results suggested that spinal NALP1 inflammasome was involved in the CCI-induced neuropathic pain and that the analgesic effect of ATL was associated with suppressing NALP1 inflammasome activation.

Introduction

Chronic neuropathic pain presents as a common disease severely disrupting the patients’ quality of life (McCarberg and Billington, 2006). Although it is a complicated condition, neuroinflammation, including glial activation and the production of proinflammatory cytokines, has been widely regarded as one important mechanism of neuropathic pain (Myers et al., 2006). The proinflammatory cytokine interleukin-1β (IL-1β) has been shown to actively participate in the pathogenesis of neuropathic pain and to induce a secondary injury cascade during the course of disease (Thacker et al., 2007). Clinically, elevated IL-1β was observed in the cerebrospinal fluid (CSF) of patients with complex regional pain syndrome (Alexander et al., 2005), and the same pathology was observed in the spinal cord of rats with neuropathic pain (Amin et al., 2012). Previous studies had demonstrated that exogenous administration of IL-1β in the CNS induced obvious pain behaviors (Sung et al., 2012). Further, blockade of IL-1 signaling has been shown to relieve nerve-injury-induced neuropathic pain (Kleibeuker et al., 2008). All these reports support a crucial role for IL-1β in the development of neuropathic pain, but the mechanisms underlying IL-1β production had not been clearly recognized, particularly in the CNS.

IL-1β is synthesized as an inactive cytoplasmic precursor and cleaved into the biologically active form by certain cysteine proteases in response to proinflammatory stimuli (Thornberry et al., 1992). The inflammasome caspase-1 platform is one of the primary pathways regulating the maturation of IL-1β (Martinon et al., 2002). Inflammasomes are groups of protein complexes that recognize diverse sets of inflammation-inducing stimuli such as pathogenic infection or tissue damage (Strowig et al., 2012). Recently, a newly discovered inflammasome family, the NAcht leucine-rich-repeat protein (NALP) inflammasome, composed of the NALP protein, caspase-1 and the adaptor protein apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (CARD) (ASC) was first found in humans and then implicated in multiple neuroinflammation-related disorders (Tschopp et al., 2003). Notably, studies have demonstrated that the NALP1 inflammasome was activated in spinal motor neurons after spinal cord injury (de Rivero Vaccari et al., 2008). In the peripheral mechanisms of complex regional pain syndrome, the NALP1 inflammasome also participated in the process of peripheral sensitization (Shi et al., 2011). However, whether the NALP1 inflammasome is activated in the spinal cord of neuropathic pain in rat has not been previously described.

Lipoxin and its exogenous analog aspirin-triggered carbon-15 epimer aspirin-triggered-15-epi-lipoxin A4 (ATL) belong to a class of eicosanoids that exhibit powerful anti-inflammatory effects and act as endogenous ‘brake signals’ in inflammation reactions (Serhan, 2005, Serhan et al., 2008). In the rat model of neuropathic pain and cancer-induced bone pain, intrathecal administration of lipoxin reduced the elevated mRNA level of IL-1β, and pain behaviors were significantly relieved (Svensson et al., 2007, Hu et al., 2012). According to our previous research, ATL showed the longest and most potent analgesic effect compared to the two structural isomers of lipoxins, lipoxin A4 and lipoxin B4 (Hu et al., 2012). In this study, we aimed to investigate whether intrathecal administration of ATL would inhibit NALP inflammasome activation and the subsequent IL-1β maturation in the spinal cords of rats with chronic constriction injury (CCI)-induced neuropathic pain.

The present study found that the spinal inflammasome-caspase-1 pathway was activated in the development of CCI neuropathic pain and inhibiting the product of activated inflammasome significantly attenuated the CCI pain behaviors. Moreover, the activated NALP1 inflammasome was found in both dorsal astrocytes and neurons, especially in the superficial laminae of the spinal dorsal horn. Finally, intrathecal administration of ATL relieved CCI-induced thermal pain behaviors and inhibited the activation of the NALP1 inflammasome, providing a new therapeutic target for neuropathic pain.

Section snippets

Animals

Experiments were performed on adult (180–200 g) male Sprague–Dawley rats. Animals were obtained from the Experimental Animal Center, Shanghai Institutes for Biological Science, Chinese Academy of Sciences. They were housed under a 12/12-h light/dark cycle at a room temperature of 23 ± 1 °C with food and water ad libitum. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Ethical Issues of the International

CCI induced pain-like behavior in rats

In the Hargreaves test, CCI on rats’ right hind limbs provoked a significant decrease in the PWL of the ipsilateral hind paws from day 3 after the sciatic nerve injury that was stably sustained for 1 month (Fig. 1A, B). It also induced a profound decrease in the mechanical threshold over the same timeframe (Fig. 1C, D). However, there were no significant differences in the above pain behaviors between normal and sham surgery rats. Collectively, these data suggest that rats subjected to the CCI

Discussion

In our study, we observed for the first time that the NALP1 inflammasome system participated in the development of neuropathic pain. CCI initiated the activation of the NALP1 inflammasome, leading to the cleavage of pro-caspase-1, upregulation of the adaptor protein ASC and the maturation of IL-1β. The components of the NALP1 inflammasome complex were visible in the astrocytes and neurons of the spinal cord dorsal horn. Repeated intrathecal administration of ATL to the CCI rats significantly

Author contributions

Qian Li carried out the major part of the study. Qian Li and Zhi-Fu Wang performed the animal surgery and the behavioral tests. Qian Li and Yu Tian carried out the immunoprecipitation study. Qian Li drafted the manuscript. Shen-Bin Liu, Wen-Li Mi and Hong-Jian Ma carried out part of the immunofluorescence study. Gen-Cheng Wu, Jun Wang and Jin Yu revised the manuscript. Yan-Qing Wang conceived and designed the study. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the National Key Basic Research program of China (2013CB531906), the National Natural Science Fund of China (30970975, 31000495, 81171045, 81072875, 81371247 and 31121061), the Excellent Doctoral Graduate Research Program of Fudan University and the Doctoral Fund of the Ministry of Education of China (Nos. 20100071120046, 20100071120042).

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