Research ArticleInhibition of Nerve Growth Factor Signaling Alleviates Repeated Dural Stimulation-induced Hyperalgesia in Rats
Introduction
Chronic migraine (CM) is a disabling neurologic condition that affects approximately 0.5–2% of the general population. Patients with CM have headaches at least 15 days a month, with at least eight days a month, their headaches and associated symptoms meet the diagnostic criteria for migraines (Schwedt, 2014, Paemeleire et al., 2015). Activation and sensitization of the trigeminovascular system are the major mechanisms of CM pathogenesis (Moskowitz, 2008, Kunkler et al., 2015). Peripheral sensitization occurs in the neuronal elements of the trigeminal ganglion (TG), while central sensitization mainly appears in the second-order neurons of the trigeminal nucleus caudalis (TNC) (Vécsei et al., 2015). Cutaneous hyperalgesia presents frequently in CM patients (Benatto et al., 2017) and is considered a risk factor for the chronicity of migraine and a clinical marker for central sensitization (Louter et al., 2013). Therefore, knowledge of how to relieve cutaneous hyperalgesia is essential for CM treatment.
Nerve growth factor (NGF) is an important signaling molecule for transmitting pain, and its signaling is considered an alternative therapeutic strategy (Suzuki et al., 2016, Majuta et al., 2017). NGF has been associated with mechanical sensitization of peripheral nociception, and the allodynia and hyperalgesia induced by NGF injection may persist for days (Wong et al., 2014, Martins et al., 2017). NGF also plays a key role in chronic pain maintenance. Clinical studies have reported that CM patients show elevated NGF in both plasma and cerebrospinal fluid (Jang et al., 2011, van Dongen et al., 2017). Based on the above findings, we speculated that NGF may be involved in cutaneous hyperalgesia of CM. NGF participates in the nociception of TG neurons both in vitro and vivo, and NGF-neutralizing antibody can counteract these effects (D'Arco et al., 2007). However, little is known about the role of NGF in the TNC site of CM. Therefore, this article explored the expression of NGF in the TNC and the role of NGF signaling pathways in the cutaneous hyperalgesia of CM.
Previous studies have reported that NGF can induce hyperalgesia by regulating the expression of functional proteins, including neurotransmitters, transient receptor potential vanilloid 1 and acid-sensing ion channels (ASICs) (Mamet et al., 2002, Pezet and McMahon, 2006). ASICs are neuronal voltage-insensitive cationic channels, activated by increasing concentrations of extracellular protons. There are six subunits of ASICs: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. The associations of different subunits form homo- or heteromeric functional ASICs (Cheng et al., 2018). ASICs, particularly ASIC3, are closely associated with migraine (Yan et al., 2011, Dussor, 2015). Decreased meningeal pH activates ASIC3, mediating dural afferent activation and migraine-related pain behaviors (Yan et al., 2013). Pretreatment with the ASIC3 inhibitor APTEx2 represses the secretion of calcitonin gene-related peptide (CGRP) from trigeminal neurons and thus may serve as an antimigraine therapy (Durham and Masterson, 2013). Our previous study found that repeated inflammatory soup (IS) dural stimulation elevated ASIC3 mRNA and protein levels at the TNC site in an animal model of migraine. In addition, APTEx2 administration attenuated tactile allodynia and decreased the expression of GRPP and c-Fos in the TNC, whereas the ASIC3 activator 2-guanidine-4-methylquinazoline exacerbated these effects (Wang et al., 2018). Protein kinase C (PKC) is a downstream signaling molecule of NGF. In the periphery, blocking PKC activity prevents NGF-mediated sensitization of nociceptive sensory neurons to painful stimulation (Bonnington and McNaughton, 2003). As a multifunctional cellular kinase, PKC increases native ASIC3-like currents in cultured dorsal root ganglion neurons (Deval et al., 2004), which suggests that there may be a regulatory relationship between PKC and ASIC3. Therefore, we hypothesized that ASIC3 is regulated by NGF through PKC in the TNC and that an NGF/PKC/ASIC3 pathway is involved in the cutaneous hyperalgesia of CM.
In this paper, we investigated the role of NGF in ASIC3 expression and repeated IS infusion-induced cutaneous hyperalgesia. NGF expression was significantly elevated in the TNC of CM rats. Moreover, anti-NGF-neutralizing antibody (anti-NGF) alleviated IS-induced cutaneous hyperalgesia and repressed the expression of PKC, ASIC3, CGRP and c-Fos in the TNC. Thus, NGF signaling might be a potential therapeutic target for CM.
Section snippets
Animals
A total of 94 male adult Sprague–Dawley rats (250–300 g) were obtained from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The experimental groups are shown in Table 1. The experiments were approved by the Ethics Committee of Chongqing Medical University. All experiments were conducted in accordance with the National Institutes of Health guide for the Laboratory animals (NIH Publications No. 80-23, revised 1978). Since a pain model was going to be established
Repeated IS infusions induced cutaneous hyperalgesia
We used a von Frey monofilament to determine the mechanical thresholds of the periorbital region and the hind paw of rats after dural infusions of IS (CM group) or PBS (Sham group). Compared with Sham group, the mechanical thresholds of the periorbital region and the hind paw were significantly decreased in the CM group. The threshold of the periorbital region decreased markedly beginning at the second day, and the mechanical threshold of the hind paw decreased significantly beginning at the
Discussion
The present study investigated the effect of NGF on ASIC3 expression in the TNC and the role of this signaling in cutaneous hyperalgesia induced by repeated IS infusions. First, after seven days of IS infusions, the pain thresholds of the periorbital region and hind paw in CM rats were significantly decreased, which indicated that IS infusions induced cutaneous hyperalgesia. Next, we found increased NGF expression in the TNC of the CM group rats. In addition, NGF was mainly expressed in the
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81671093 and No. 81500957) and the District Science and Technology Projects of Yuzhong Chongqing (No. 20160107). The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Lixue Chen designed and monitored this work. Huiru Zhou performed the experiments, analyzed data, prepared figures, and drafted the manuscript. All authors contributed to
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