The contribution of local ETA
The contribution of local ETA and ETB receptors towards heat hyperalgesia induced by inoculation of XC tumor Amyloid β-Peptide (1-42) pathway in the paw of mice has been demonstrated by Baamonde and colleagues (Baamonde et al., 2004). Furthermore, using neuropathic and inflammatory nociception models, several studies have shown a significant effect of local injection of ETA or ETB receptor antagonists in controlling heat hyperalgesia (Baamonde et al., 2004; Fattori et al., 2017; Khodorova, Montmayeur, & Strichartz, 2009; Werner et al., 2010). More specifically in the orofacial region, previous reports also demonstrated the participation of peripheral ETA and ETB receptors in the development of heat hyperalgesia in a model of trigeminal neuropathic pain (Chichorro et al., 2009). Likewise, herein we demonstrated that peripheral ETA and ETB endothelin receptors also seem to be involved in tumor-induced facial heat hyperalgesia. Thus, heat hyperalgesia in conditions such as neuropathy and cancer appears to be mediated by peripheral ETA and ETB receptors. These findings raise another possibility for the lack of efficacy for the selective ETA and ETB receptor antagonists in contrast to the antinociceptive effect achieved with the dual ETA/ETB endothelin receptors antagonist, bosentan. In accordance to this hypothesis, it would be necessary the blockade of both endothelin receptors to interfere with heat hyperalgesia an spontaneous grooming associated with facial cancer. In line with this idea, the co-administration of BQ-123 with BQ788 results in a tendency to decrease the facial grooming. However, this question deserves further investigation, since most of the mechanisms related to endothelin nociceptive effects are unknown. However, it has been shown that activation of ETA receptors expressed in trigeminal peripheral sensory fibers cause sensitization of TRPV1 receptors resulting in heat hyperalgesia (Plant, Zollner, Mousa, & Oksche, 2006, 2007). Likewise, ETB receptors have been shown to be expressed by non-peptidergic C fibers and satellite glia cells of the trigeminal system, but the mechanisms underlying their hyperalgesic effects are currently unknown (Brandli et al., 1996; Chichorro et al., 2009, 2010; Kitano et al., 1998). The inoculation of tumor cells in the facial region of rats also increases spontaneous facial grooming behavior, which is suggestive of spontaneous nociception in rodents (Akiyama, Carstens, & Carstens, 2010; Hidaka et al., 2011; Kopruszinski et al., 2018; Ono et al., 2009; Sago et al., 2012; Spradley, Davoodi, Carstens, & Carstens, 2012). Interestingly, there are preclinical and clinical indicatives of the importance of endothelin-1 for the promotion and maintenance of spontaneous nociception (Gokin et al., 2001; Hans, Schmidt, & Strichartz, 2009; Pickering et al., 2007, 2008; Smith, Haymond, Smith, & Sweitzer, 2014). In addition, preclinical studies using different cancer models reported elevated endothelin levels in animals that demonstrate evoked hypersensitivity or signs of spontaneous nociception, highlighting the contribution of endothelin receptors to the development of these aspects of cancer pain (Awano, Dawson, Hunter, Turner, & Usmani, 2006; Kandalaft, Facciabene, Buckanovich, & Coukos, 2009; Mankapure, Barpande, Bhavthankar, & Mandale, 2015; McKenzie et al., 2014; Pickering et al., 2008; Schmidt et al., 2007). In line with these observations, our group has shown that systemic treatments with bosentan and morphine markedly reduced the incidence of spontaneous grooming associated with the facial tumor (Kopruszinski et al., 2018). The present study evidenced the effectiveness of local bosentan treatment in controlling tumor-induced increased spontaneous grooming. Thus, processing of spontaneous pain-like behavior appears to be mediated by peripheral (local) ETA and ETB receptors. In order to further explore the role of peripheral endothelins on spontaneous nociception, we also evaluated the effect of local treatment with bosentan, lidocaine and morphine in tumor-induced ongoing nociception, through the induction of conditioned place preference (CPP). Our data failed to reveal the occurrence of CPP induction in tumor-bearing rats after local treatments. On the other hand, previous studies have already revealed the efficacy of systemic treatment with bosentan (Kopruszinski et al., 2018) and morphine (Kopruszinski et al., 2018; Remeniuk et al., 2015) in controlling tumor-induced ongoing nociception, observed by significant CPP for the drug-paired chamber. We speculate that the tumor-induced increased spontaneous grooming might represent a phasic pain component of spontaneous nociception, whereas tumor-induced ongoing pain may constitute a tonic component of spontaneous nociception. These different components of pain also indicate distinct mechanisms. The results of the current study might suggest that the reduction of peripheral sensitization by locally injected drugs is not sufficient to control tumor-induced ongoing nociception, which require additional central mechanisms. Corroborating this hypothesis, some authors suggested that the mechanism related to the relief of ongoing nociception in the CPP test is through the activation of the corticolimbic system (Navratilova, Xie, King, & Porreca, 2013; Porreca & Navratilova, 2017). Moreover, Sago et al. (2012) suggested the existence of a microglia-dependent activation pathway for the development of ongoing nociception induced by the tumor (Sago et al., 2012). Additionally, preclinical studies demonstrate that some tumors can promote nerve sprouting and neuronal reorganization, which seems to contribute directly to the emergence and maintenance of changes related to ongoing nociception (Jimenez Andrade & Mantyh, 2010; Mantyh, Clohisy, Koltzenburg, & Hunt, 2002, 2010).