Wnt5a mediates chronic post-thoracotomy pain by regulating non-canonical pathways, nerve regeneration, and inflammation in rats
Graphical abstract
Introduction
Management strategies for chronic pain after surgery, particularly amputation, breast surgery, and thoracotomy, are inadequate considering the high incidence of such chronic pain and insensitivity to typical therapies [1, 2]. Chronic post-thoracotomy pain (CPTP), which recurs or persists along a thoracotomy incision at least 2 months following the surgical procedure [3], with a high incidence of CPTP (50%) for 3–6 postoperative months [4], has become a major research focus in the past two decades. Surgical-associated intercostal nerve injury is believed to be a dominant factor in the pathogenesis of CPTP, as demonstrated by intraoperative electrophysiology examinations [5] and the high proportion of neuropathic pain in CPTP [6, 7]. However, strategies aimed at protecting the intercostal nerve do not always prevent CPTP, indicating that complex additional factors are involved. Thus, additional research into the effective management CPTP pathogenesis are needed.
In a pilot study [8], we showed that Wnt5a inhibition alleviates CPTP. This finding was consistent data showing that levels of circulating microRNAs targeting Wnt pathway members in rodent models of both neuropathic pain and inflammatory pain were changed, suggesting Wnt as a possible biomarker for chronic pain [9]. Moreover, Wnts are involved in both the induction and persistence of neuropathic pain [10, 11], accompanied with a rapid-onset and long-lasting upregulation of pro-inflammatory factors and calcium signals in the spinal cord [10]. Wnts also play a role in cancer pain by activating non-canonical Wnt pathways [12]. As a secreted glycoprotein, Wnt5a acts mainly via the non-canonical planar cell polarity, or Ca2+ pathway, rather than via the canonical β-catenin pathway [13], whose effects are often opposite [14]. The downstream cellular signals of Wnt5a in CPTP remain to be elucidated.
Quite recently, it was reported that the inability of regenerating nerves to re-innervate target tissues results in the persistence of neuropathic pain [15], suggesting a crucial role of nerve regeneration in the condition. As critical ligands in maintaining neuronal functions and tissue morphogenesis, dysregulated Wnts have been identified in various degenerative and inflammatory central nervous system disorders [13, 16, 17]. Thus, we further tested the involvement of nerve regeneration in the maintenance of CPTP and its regulation by Wnt5a. Additionally, Wnt expression in the immune system and in immune-like cells of the central nervous system suggested its involvement in inflammation-related damage or repair [13]. Similarly, we found that the activated pro-inflammatory response in CPTP could be regulated by Wnt5a [8]. Interestingly, patients with more serious acute post-thoracotomy pain were more likely to develop chronic pain [18], indicating that alterations in the acute phase may contribute to the development of CPTP. Moreover, in our retrospective study of CPTP, we found that the difference between pre- and post-operative day 2 white blood cell counts could predict the occurrence of CPTP [19]. Thus, we further examined the dynamic changes of systemic inflammation and neuroinflammation to determine these changes are correlated with CPTP development.
In this study, we focused on the elusive mechanism of Wnt5a in the induction and persistence of CPTP, and its regulation of Wnt pathways, nerve regeneration, and inflammation.
Section snippets
Animals
Adult male Sprague-Dawley rats (250–350 g, Experimental Animal Center of the Chinese Academy of Medical Sciences, Beijing, China) were used in this study. Rats were housed at a temperature of 22–24 °C under a 12 h light-dark cycle. All animal protocols were approved by the Institutional Animal Care and Use Committee in the Chinese Academy of Medical Sciences. The procedures were performed in accordance to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Rats were
CPTP induced time-course activation of Wnt5a and Ror2 in the DRG
Rats were divided into the naïve group (n = 10), sham group (n = 10), and model group (n = 20), which was further divided into the CPTP group and non-CPTP group according to the pain state observed on POD 21. Rats with an MWT ≦4 g on POD 21 were assigned to the CPTP group (n = 11), and the remaining rats were assigned to the non-CPTP group (n = 9). The pain incidence in all our model group was consistent with that reported in previous studies [20, 21]. The MWT in the CPTP group showed a sharp
Discussion
Emerging evidence has shown that Wnts play a critical role in the pathogenesis of neuropathic pain [10], the persistence of inflammatory pain [10], and the pathogenesis of human immunodeficiency virus-induced pain [28]. In this study, we found that Wnt5a was activated on POD 7 and upregulated over time in CPTP. Inhibiting Wnt5a by antagonizing its binding site significantly reduced pain-related behavior. Moreover, early intervention during the preliminary and acute phases resulted in enhanced
Conclusions
Collectively, we found that the activated Wnt5a/Ca2+ and Wnt5a/planar cell polarity pathways, along with increased axonal regeneration and sympathetic sprouting (which could be regulated by Wnt5a), are involved in CPTP persistence. The accompanying activation of inflammation modulated by Wnt5a may play a role in CPTP induction and persistence. Further, this study suggests that early (but not late) therapy targeting Wnt5a may represent an effective treatment for CPTP.
Funding
This study was supported by grants from National Natural Science Foundation of China (NO: 81671098).
Competing interests
The authors declare no competing interests.
Author contributions
YH designed the experiments and reviewed the manuscript. AF carried out the thoracotomy, intrathecal catheterization and injection, western blot, RT-PCR, immunohistochemistry, ELISA and prepared the manuscript. LS, LX and WC conducted the pain behavior measurements, analyzed the data, and reviewed the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
We thank Dr. Xin Zhao for supplying the animal ventilator. We also thank Professor Chao Ma and post-doctoral fellow Fan Liu for helpful comments on the experiments.
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