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Blockade of peripheral endothelin receptors abolishes heat hyperalgesia and spontaneous nociceptive behavior in a rat model of facial cancer
Archives of Oral Biology 97 (2019) 231–237
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Blockade of peripheral endothelin receptors abolishes heat hyperalgesia and spontaneous nociceptive behavior in a rat model of facial cancer Caroline Machado Kopruszinskia, Juliana Geremias Chichorroa a b
⁎,1
T
, Renata Cristiane dos Reisa, Giles Alexander Raeb,
Department of Pharmacology, Biological Sciences Section, Federal University of Parana, Curitiba, Brazil Department of Pharmacology, Biological Sciences Center, Federal University of Santa, Catarina, Florianopolis, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Walker-256 tumor cells Evoked nociception Facial grooming Endothelins ETA receptor ETB receptor
Objective: To improve understanding of the pathophysiology of cancer-induced facial nociception, by evaluating the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception in a rat model of facial cancer. Design: The study was conducted using 396 rats. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rats’ right vibrissal pad. Facial heat hyperalgesia and spontaneous grooming were assessed on day 6, while the conditioned place preference (CPP) test was performed on days 3–6 after tumor cells inoculation. Rats received local injections of the non-peptidic dual ETA/ETB endothelin receptors antagonist, bosentan (10 and 30 μg/50 μL), single or combined injections of peptidic ETA and ETB endothelin receptors antagonists (BQ-123 and BQ-788, at 20 ug/50 μL, each), or of lidocaine (1 mg/50 μl) and morphine (30 μg/ 50 μL). Results: Bosentan, lidocaine and morphine local treatment all attenuated tumor-induced heat hyperalgesia (p < 0.05) and spontaneous facial grooming (p < 0.05). However, BQ-123 and BQ-788 did not modify tumorinduced heat hyperalgesia or the spontaneous facial grooming (p > 0.05). Whether this difference in effectiveness is due to receptor affinity or to pharmacokinetic factors still needs to be explored. Local injection of bosentan, lidocaine or morphine failed to control ongoing nociception, as evidenced by the absence of CPP in tumor-bearing rats (p > 0.05). Conclusion: Endothelins, acting through peripheral ETA and ETB receptors, may play a significant role on the development of heat hyperalgesia and increased spontaneous grooming associated to facial cancer in rats.
1. Introduction Pain is frequently the first clinical symptom of head and neck carcinoma. Indeed, pain is the initial reason leading ∼70% of cancer patients to seek professional assistance (Lam & Schmidt, 2011; Rettig & D’Souza, 2015; Schmidt, 2014). Clinically, cancer-induced facial pain can be evoked or occurs spontaneously and its intensity is characterized as moderate to severe (Lam & Schmidt, 2011; Viet & Schmidt, 2012). The current treatment protocols follow the analgesic ladder recommendation of the World Health Organization (WHO) for cancer pain management (Mercadante & Fulfaro, 2005; Mercadante & Giarratano, 2013; Mercadante, 2010). However, studies demonstrated that a significant proportion of patients have unsatisfactory pain control and present many side effects that markedly reduce their quality of life
(Dy et al., 2008; Foley, 2004). Thus, a better understanding of the pathophysiology of cancer-induced facial pain and the improvement of therapeutic strategies for pain control are clearly warranted (Lam & Schmidt, 2011; Schmidt, 2014, 2015). There is mounting evidence for the involvement of endothelins in many processes related to facial cancer, including pain (McKenzie, Hinsley, Hunter, & Lambert, 2014; Pickering, Jay Gupta, Quang, Jordan, & Schmidt, 2008; Quang & Schmidt, 2010b; Russo et al., 2010). Increased serum levels of endothelin-1 (ET-1) have been detected in patients with facial cancer (Pickering, Jordan, & Schmidt, 2007), as well as in tumor-bearing animals, and these alterations have been associated with tumor progression and the development of spontaneous and evoked nociception (Connelly & Schmidt, 2004; Fujita, Andoh, Saiki, & Kuraishi, 2008; Lam & Schmidt, 2011; Pickering et al., 2008;
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Corresponding author at: University of Arizona, College of Medicine, Department of Pharmacology, Tucson, AZ, USA. E-mail address: [email protected] (C.M. Kopruszinski). 1 Present address: University of Arizona, College of Medicine, Department of Pharmacology, Tucson, AZ, USA. https://doi.org/10.1016/j.archoralbio.2018.10.038 Received 15 July 2018; Received in revised form 30 October 2018; Accepted 31 October 2018 0003-9969/ © 2018 Published by Elsevier Ltd.
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Aldrich, San Francisco, USA) at 30 μg/50 μL, the peptidic endothelin ETA and ETB receptor antagonists BQ-123 and BQ-788 (American Peptides, San Francisco, USA) were injected alone or in combination at 20 μg/50 μL, whereas the non-peptidic dual endothelin ETA and ETB receptor antagonist bosentan monohydrate (Actelion Pharmaceuticals, Allschwill, Switzerland) was administered at 10 or 30 μg/50 μL. Controls animals received a 50 μL injection of saline. The choice of drug doses were based on the literature (Chichorro, Zampronio, Cabrini, Franco, & Rae, 2009, 2010; Kopruszinski et al., 2018; Remeniuk et al., 2015).
Schmidt et al., 2007; Yan, Peng, & Huang, 2015). Moreover, preclinical studies in different cancer models have demonstrated the efficacy of endothelin receptor blockade on tumor-induced sensory changes. These studies highlight that the endothelin system is implicated in tumor-induced sensory changes associated with cancer cells inoculation (Fujita et al., 2008; Hamamoto, Khasabov, Cain, & Simone, 2008; Quang & Schmidt, 2010a; Schmidt et al., 2007). We recently demonstrated that systemic blockade of endothelin ETA and ETB receptors abolished tumor-induced evoked and ongoing nociception in a rat model of facial carcinoma. It was also shown that systemic treatment with an endothelin receptor antagonist potentiated morphine-induced antinociception, indicating that endothelin receptor blockade may represent an improved new treatment strategy for patients with head and neck cancer pain (Kopruszinski et al., 2018). However, the involvement of the peripheral endothelin system in evoked and ongoing nociception related to facial cancer has not yet been characterized. In light of these considerations, this study examined the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception.
2.4. Heat Stimulation
2. Material and methods
Heat sensory thresholds of the vibrissal pad area were assessed as previously reported (Chichorro et al., 2009). Briefly, each animal was lightly restrained by the experimenter while a ∼50 °C radiant heat source was positioned at 1 cm from the surface of the right vibrissal pad. The response latency to display either head withdrawal or vigorous flicking of the snout was recorded and adopted as a behavioral index of heat sensitivity. A 20 s cut-off time was used to prevent tissue damage.
2.1. Animals
2.5. Spontaneous grooming evaluation
This study was conducted on male Wistar rats weighing 180–220 g. Animals were housed in a temperature-controlled room, under 12 h dark/light circadian cycle, with free access to chow and tap water. All experimental procedures were performed in accordance with the ARRIVE guidelines, with the ethical guidelines of the International Association for the Study of Pain regulations on animal welfare and the National Institutes of Health guide for the care and use of laboratory animals. The experiments were previously approved by Federal University of Parana - Institutional Committee on the Ethical Use of Animals (authorization # 645). This study used 396 rats total, 366 animals for all the behaviors experiments and 30 animals for in vivo maintenance of the tumor cells. To determine the sample size is a requirement of the Committee on the Ethical Use of Animals of our institution before the approval of the experimental Protocols. We have determined the sample size by using the GPower 3.1 software, which, considering a significance level of 5%, estimated a minimum of 5 rats for experiments assessing thermal hyperalgesia, 11 rats for the conditioned place preference evaluation and 17 for the analysis of facial grooming. All efforts were made to diminish the number of animals used and their suffering along the study.
Evaluation of spontaneous facial grooming was performed as previously described (Kopruszinski et al., 2018). In brief, rats were submitted to individual evaluation of time spent performing spontaneous facial grooming across a 10-minutes session conducted between 8:0011:00 h. Facial grooming behavior was defined as bilateral cleaning of the face, at the vibrissae region, with the front paws (Hidaka et al., 2011). 2.6. Conditioned place preference Evaluation of ongoing/tonic nociception relief using the conditioned place preference (CPP) paradigm was performed as previously described (King et al., 2009). The CPP apparatus consist of three chambers designated as two conditioning chambers, with each compartment measuring 36 × 25 × 40 cm, with distinct visual and tactile cues, i.e. different-patterned walls (one chamber with black walls and the other one with striped walls) and texturally distinct floors (black chamber with smooth floor and stripped chamber with textured floor), respectively, separated by a neutral chamber measuring 36 × 25 × 10 cm, that was brightly lit (LED light 100 lx). The CPP paradigm consists in three steps, pre-conditioning or baseline, conditioning and test. The pre-conditioning step, defined as the first day of CPP paradigm, animals were located into the neutral chamber with free access to all chambers, and left to explore the chambers for 15 min. Exploratory behavior was filmed and recorded, with the baseline of the time-spent in each chamber analyzed. To avoid preference for one of the chambers on the baseline, rats that spent more than 80% or less than 20% in one of the conditioning chambers were removed from the study. Drug pairing was assigned to the conditioning chambers using an unbiased CPP design, with equivalent mean times across the two conditioning chambers. Drug assignment was counterbalanced across the conditioning chambers according to the amount of the time spent in each chamber during the baseline step, i,e, half of the animals were paired to the drug treatment in one of the conditioning chambers for all groups and the other half were paired to the drug treatment on the opposite conditioning chamber. A two conditioning days CPP protocol was chosen based on the drug route treatment, whereby the conditioning step occurred across two consecutive days, as detailed below. On the test day, rats were once again placed into the neutral chamber with free access to all chambers, and allowed to explore the apparatus for 15 min. Time spent in each compartment was analyzed to determine whether drug treatment produced CPP, indicated by an increased time
2.2. Maintenance and inoculation of the tumor cells To induce facial carcinoma in rats, Walker-256B lineage cells were used as previously described (Kopruszinski et al., 2018). The cells were maintained in vivo by weekly intraperitoneal (ip) passages. To induce the facial cancer, the animals were anesthetized with a solution of xylazine/ketamine (7.5/60 mg/kg, respectively, ip) and injected subcutaneously (sc) with a suspension of 2 × 106 Walker-256B cells in 100 μL into the right vibrissal pad. Control animals (sham) were similarly treated with 100 μL of vehicle (phosphate-buffered saline, PBS). The day of inoculation of the Walker-256B cells was considered day 0. From the total animals used for the behavior experiments, less than 5% did not develop tumor at the facial region. These animals were excluded of the experiments. 2.3. Drugs All drugs evaluated in the present study were diluted in 0.9% NaCl (i.e. saline), and administered locally, through sc injection into the rat’s right upper lip. Lidocaine hydrochloride (Cristália, Sao Paulo, Brazil) was administered at 1 mg/50 μL, morphine hydrochloride (Sigma 232
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in seconds spent in the drug-paired chamber as compared to pre-conditioning time and post-conditioning vehicle-paired chamber time, with positives scores indicating preference. 2.7. Behavioral analysis protocols The effects of local treatment with lidocaine, morphine (used as control drugs) or with endothelin receptor antagonists on tumor-induced heat hyperalgesia was assessed in different cohorts of rats, on day 6 post-inoculation. A total of 120 rats were used for this experiment. Baseline responsiveness to heat stimulation was evaluated solely on the right (ipsilateral) vibrissal pad prior (BL) to inoculation of tumor cells and again on day 6 (D6) to verify tumor-induced heat hyperalgesia. Following confirmation of heat hyperalgesia on day 6, bosentan, BQ123, BQ-788, BQ-123 plus BQ-788 in association, lidocaine, morphine or vehicle were all administered locally into the right upper lip. Heat hyperalgesia was re-evaluated at 30 min and again every hour up to 4 h after drug or vehicle treatment. The effect of the same treatments was assessed 30 min after their administration on tumor-induced increased spontaneous grooming in different cohorts of animals on day 6 post tumor-inoculation. A total of 213 rats were used for this experiment. In addition, CPP to local bosentan, lidocaine or morphine treatments was evaluated in separate cohorts of rats, on days 3–6 post tumor-inoculation. The pre-conditioning day occurred on day 3 after tumor cell inoculation as described above. The two conditioning days (days 4 and 5 after tumor cells inoculation) were divided in morning and afternoon sections. In the morning, rats were injected locally with vehicle and immediately confined to the appropriate pairing chamber for 30 min. Four hours later (in the afternoon), the same animals were injected locally with bosentan, lidocaine or morphine and immediately confined into the opposite pairing chamber for 30 min. The test day took place on day 6 after tumor cell inoculation. A total of 33 rats were used for this experiment. The timeline of the experimental procedures is illustrated in Fig. 1.
Fig. 1. Experimental protocols timeline. A. Heat stimulation: on day 0, baseline to facial heat stimulation was collected followed by the inoculation of tumor cells into animal’s right vibrissal pad. On day 6 after inoculation, facial heat stimulation measurement was performed to confirm the development of heat hyperalgesia. Treatment was performed subcutaneously (right upper lip) and facial heat stimulation was assed hourly. B. Spontaneous facial grooming: on day 0, animals were submitted to the inoculation of tumor cells into animal’s right vibrissal pad. On day 6 after inoculation, treatment was performed subcutaneously (right upper lip) and spontaneous facial grooming was assessed for 10 min after the treatment. C. Conditioned place preference (CPP): on day 0, animals were submitted to the inoculation of tumor cells into animal’s right vibrissae pad. On day 3, drug-free animals were placed into CPP apparatus with free access to all three chambers for baseline collection. On days 4 and 5, rats were exposed to two conditionings days. In the mornings (AM), animals were treated with vehicle and conditioned to a designed chamber, with no access to the other chambers. In the afternoons (PM), animals were treated with drug and conditioned to the opposite chamber to the morning section, also with no access to the other chambers. On day 6, drug-free animals were submitted to the test day, with free access to all three chambers, for the evaluation of conditioned place preference.
2.8. Statistical analysis Data are presented as mean ± S.E.M. and n represent the number of rats analyzed. Heat hyperalgesia time-course data for drug effects were analyzed using two-way ANOVA followed by Tukey post-hoc tests. For spontaneous grooming and CPP data, the effects of treatment were analyzed by two-way ANOVA followed by Tukey post-hoc test between baseline (pre-conditioning) and test (post-conditioning) values. All data were analyzed with GraphPad Prism® 6. A statistically difference was set to a probability level of 0.05. Error bars are S.E.M. 3. Results 3.1. Effect of local endothelin receptor blockade on tumor-induced heat hyperalgesia
peptidic dual ETA/ETB endothelin receptor antagonist bosentan attenuated the tumor-induced heat hyperalgesia for one hour, starting within 30 min of treatment, at both doses tested (Fig. 2C, p < 0.05 versus sham rats treated with vehicle and p < 0.05 versus tumorbearing rats treated with vehicle). Sham animals treated with vehicle or bosentan did not display any difference in response latencies at any time point (Fig. 2C, p > 0.05).
Tumor-bearing rats developed heat hyperalgesia (i.e. a decrease in response latency relative to baseline), which was attenuated by local control treatments, lidocaine and morphine. Both treatments significantly reduced tumor-induced heat hyperalgesia within 30 min of treatment, and the response latencies returned to pre-lidocaine and premorphine values by 1 and 2 h post-administration, respectively (Fig. 2A, p < 0.05 versus sham rats treated with vehicle and p < 0.05 verustumor-bearing rats treated with vehicle). Sham animals treated with lidocaine or morphine did not show any difference in response latencies when compared to vehicle-treated controls at any time-point (Fig. 2A, p > 0.05). On day 6 post inoculation, local treatment with BQ-123 or BQ-788, either alone or in combination, failed to modify the heat hyperalgesia induced by tumor cell inoculation (Fig. 2B, p < 0.05 versus sham rats treated with vehicle). In contrast, similar treatment with the non-
3.2. Effects of local endothelin receptor blockade on tumor-induced increase in facial grooming On day 6 post tumor cell inoculation, tumor-bearing rats displayed increased spontaneous grooming relative to sham controls (Fig. 3A, p < 0.01 versus sham rats treated with vehicle). Local administration of the control drugs, lidocaine or morphine blocked the increase in spontaneous grooming of tumor bearing rats to values equivalent to those shown by sham controls (Fig. 3A, p < 0.05 versus tumor-bearing 233
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Fig. 2. Effect of local administration of lidocaine, morphine and endothelin receptor antagonists on tumor-induced facial heat hyperalgesia. A. Local treatment with lidocaine and morphine reduced heat hyperalgesia in tumorbearing rats on day 6 post inoculation by 30 min after administration (*p < 0.05 versus Sham-Vehicle and #p < 0.05 versus Tumor-Vehicle, n=58). B. Local administration of BQ-123 and BQ-788, either alone or in combination, failed to modify tumor-induced heat hyperalgesia (*p < 0.05 versus Sham-Vehicle, n=6-10). C. Local treatment with bosentan, at both doses, reduced heat hyperalgesia in tumor-bearing rats by 30 min post administration (*p < 0.05 versus Sham-Vehicle and #p < 0.05 versus Tumor-Vehicle, n=612). Results are presented as mean ± S.E.M. and statistical analysis was assessed by two-way ANOVA followed by Tukey test.
modify the tumor-induced increase in grooming behavior when tested at the lower dose of 10 μg (Fig. 3C, p > 0.05versus tumor-bearing rats treated with vehicle). Nonetheless, local bosentan treatment at the higher dose of 30 μg effectively blocked tumor-induced increase in grooming, with the time spent grooming equivalent to that of sham controls (Fig. 3C, p < 0.001 versus tumor-bearing rats treated with vehicle and p > 0.05 versus sham rats treated with vehicle). Also, bosentan at 30 μg did not modify the grooming behavior of sham rats (Fig. 3C, p > 0.05versus sham rats treated with vehicle). 3.3. Local lidocaine, morphine and bosentan treatment failed to control ongoing nociception related to facial cancer Local treatments with bosentan (30 μg/50 μl), lidocaine (1 mg/ 50 μl) or morphine (30 μg/50 μL) failed to induce CPP on the test day (i.e. day 6 after tumor cell inoculation), as evidenced by the absence of any significant alterations in the time spent in the pairing chambers following two conditioning sessions on days 4 and 5 after inoculation. Pre-conditioning (baseline) scores measured on day 3 after inoculation did not differ between the vehicle-paired chamber and the drug-paired chamber. On the test day, local lidocaine, morphine and bosentan treatments did not increase time spent in the drug-paired chamber of tumor-bearing animals when compared to pre-conditioning scores measures (Table 1). 4. Discussion This study showed the ability of peripheral blockade of both ETA and ETB endothelin receptors in controlling the facial heat hyperalgesia and increased facial grooming behavior in a model of facial cancer in rats. On the other hand, the current data demonstrate that peripheral dual blockade of both ETA and ETB receptors failed to modify tumorinduced ongoing facial nociception. These data suggest that peripheral ETA and ETB receptors mediate responses related to evoked nociception and increased grooming induced by facial cancer. We recently demonstrated that systemic treatment with the dual endothelin ETA and ETB receptor antagonist bosentan abolished tumorinduced heat hyperalgesia, increased spontaneous grooming and ongoing nociception in the same facial cancer model used in the present study (Kopruszinski et al., 2018). However, the contribution of peripheral endothelins was not investigated. Herein, it was shown that local treatment with bosentan blocked facial heat hyperalgesia and increased facial grooming in tumor-bearing rats. This effectiveness was similar to that promoted by local treatment with morphine and lidocaine, used as control drugs in this study. On the other hand, local treatments with the peptidic endothelin receptors antagonists BQ-123 and BQ-788, alone or in association, were unable to reduce the facial heat hyperalgesia or increased facial grooming of tumor-bearing rats. This ineffectiveness might be a result of increased synthesis and release of proteases by the tumor cells, resulting in the hydrolysis, and consequently, the degradation of the BQ molecules prior to receptor binding (Hardt, Lam, Dolan, & Schmidt, 2011; Schmidt, 2014). Thus, these data suggest that local both receptors contribute to the development of facial
rats treated with vehicle). Local administration of lidocaine or morphine to sham rats failed to alter grooming behavior (Fig. 3A, p > 0.05). Local administration of BQ-123 or BQ-788, either alone or in association, failed to reduce the increase in grooming behavior observed on day 6 after tumor cell inoculation (Fig. 3B, p < 0.01, p < 0.05 and p < 0.05 versus Sham-Vehicle). Likewise, bosentan also failed to 234
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Table 1 Effect of local administration of lidocaine, morphine or dual ETA/ETB endothelin receptor antagonist bosentan on tumor-induced ongoing/tonic nociception. Local treatment with lidocaine, morphine or bosentan failed to induce CPP on the test day (day 6 post tumor cell inoculation), as attested by absence of significant increases in the time spent in seconds in the drug-paired chamber in a CPP paradigm, when compared to pre-conditioning (baseline) scores measures (p > 0.05 versus BL, n = 10–12). Results are presented as mean ± SEM and statistical analysis was assessed by two-way ANOVA followed by Tukey test. Time spent in drug-paired chamber (seconds) Group
Baseline
Test
Lidocaine Morphine Bosentan
396.1 ± 40.5 403.0 ± 28 396.9 ± 29.4
453.2 ± 89.4 466.5 ± 94.8 349.8 ± 77.5
heat hyperalgesia associated to facial cancer and that dual endothelin receptor blockade with a non-peptidic drug was effective to control tumor-induced heat hyperalgesia. The contribution of local ETA and ETB receptors towards heat hyperalgesia induced by inoculation of XC tumor cells 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 BQ123 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). In addition to these hypotheses, other factors related to the pharmacokinetics of BQs and bosentan, such as their distribution profile, or related to the receptor binding affinity may account to the difference in effectiveness, but these aspects remain to be investigated. 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,
Fig. 3. Effect of local administration of lidocaine, morphine and endothelin receptor antagonists on tumor-induced facial grooming. A. Tumor inoculation increased facial grooming behavior on day 6 post inoculation. Local treatment with lidocaine or morphine reduced the facial grooming in tumor-bearing rats (**p < 0.01 versus Sham-Vehicle and #p < 0.05 versus Tumor-Vehicle, n=11-14). B. Local administration of BQ-123 and BQ-788 alone or associated failed to modify tumor-induced facial grooming (*p < 0.05 and **p < 0.01 versus Sham-Vehicle, n=8-18). C. Local treatment with bosentan reduced the facial grooming in tumor-bearing rats only at the highest dose (***p < 0.001 versus Sham-Vehicle and ###p < 0.001 versus Tumor-Vehicle, n=11-22). Results are presented as mean ± S.E.M. and statistical analysis was assessed by two-way ANOVA followed by Tukey test.
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of Higher Education Personnel (CAPES) during the study development.
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 tumorbearing 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). Altogether, the results of this study demonstrate that peripheral blockade of both ETA and ETB receptors reduces tumor-induced facial heat hyperalgesia and increased spontaneous grooming, without modifying ongoing nociception. It is possible to suggest that bosentan is acting upon both receptors present in primary sensory afferents, or is causing the blockade of ETA, and mainly ETB receptors, present in tumor cells, promoting the blockade of endothelin-1 release from these cells, which in both cases will prevent peripheral nociceptor sensitization (Barr, Kam, Khodorova, Montmayeur, & Strichartz, 2011; Khodorova et al., 2009; Schmidt, Hamamoto, Simone, & Wilcox, 2010; Schmidt, 2014; Smith et al., 2014). The current findings contribute to the knowledge of the pathophysiology of cancer-induced facial pain and may facilitate the clinical exploration of the benefits of endothelin receptor blockade as a useful adjuvant strategy for facial cancer pain management.
Conflict of interest The authors declare no conflicts of interest. Acknowledgements The group is grateful to CNPq and CAPES for their funding support. We thank Dr. Sandra Coccuzzo Sampaio (Butantan Institute, Sao Paulo, Brazil) for kindly donating the Walker-256B tumor cells. We also thank Dr. Alexandra Acco, M.Sc. Eder Gambeta and M.Sc. Larissa Galuppo for technical support. References Akiyama, T., Carstens, M. I., & Carstens, E. (2010). Spontaneous itch in the absence of hyperalgesia in a mouse hindpaw dry skin model. Neuroscience Letters, 484(1), 62–65. https://doi.org/10.1016/j.neulet.2010.08.020. Awano, S., Dawson, L. A., Hunter, A. R., Turner, A. J., & Usmani, B. A. (2006). Endothelin system in oral squamous carcinoma cells: Specific siRNA targeting of ECE-1 blocks cell proliferation. International Journal of Cancer, 118(7), 1645–1652. https://doi. org/10.1002/ijc.21525. Baamonde, A., Lastra, A., Fresno, M. F., Llames, S., Meana, A., Hidalgo, A., ... Menendez, L. (2004). 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Funding sources This study was supported by National Council for Scientific and Technological Development (CNPq), grant #477679/2012-9. The first author received PhD. scholarship by Coordination for the Improvement 236
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Blockade of peripheral endothelin receptors abolishes heat hyperalgesia and spontaneous nociceptive behavior in a rat model of facial cancer Caroline Machado Kopruszinskia, Juliana Geremias Chichorroa a b
⁎,1
T
, Renata Cristiane dos Reisa, Giles Alexander Raeb,
Department of Pharmacology, Biological Sciences Section, Federal University of Parana, Curitiba, Brazil Department of Pharmacology, Biological Sciences Center, Federal University of Santa, Catarina, Florianopolis, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Walker-256 tumor cells Evoked nociception Facial grooming Endothelins ETA receptor ETB receptor
Objective: To improve understanding of the pathophysiology of cancer-induced facial nociception, by evaluating the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception in a rat model of facial cancer. Design: The study was conducted using 396 rats. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rats’ right vibrissal pad. Facial heat hyperalgesia and spontaneous grooming were assessed on day 6, while the conditioned place preference (CPP) test was performed on days 3–6 after tumor cells inoculation. Rats received local injections of the non-peptidic dual ETA/ETB endothelin receptors antagonist, bosentan (10 and 30 μg/50 μL), single or combined injections of peptidic ETA and ETB endothelin receptors antagonists (BQ-123 and BQ-788, at 20 ug/50 μL, each), or of lidocaine (1 mg/50 μl) and morphine (30 μg/ 50 μL). Results: Bosentan, lidocaine and morphine local treatment all attenuated tumor-induced heat hyperalgesia (p < 0.05) and spontaneous facial grooming (p < 0.05). However, BQ-123 and BQ-788 did not modify tumorinduced heat hyperalgesia or the spontaneous facial grooming (p > 0.05). Whether this difference in effectiveness is due to receptor affinity or to pharmacokinetic factors still needs to be explored. Local injection of bosentan, lidocaine or morphine failed to control ongoing nociception, as evidenced by the absence of CPP in tumor-bearing rats (p > 0.05). Conclusion: Endothelins, acting through peripheral ETA and ETB receptors, may play a significant role on the development of heat hyperalgesia and increased spontaneous grooming associated to facial cancer in rats.
1. Introduction Pain is frequently the first clinical symptom of head and neck carcinoma. Indeed, pain is the initial reason leading ∼70% of cancer patients to seek professional assistance (Lam & Schmidt, 2011; Rettig & D’Souza, 2015; Schmidt, 2014). Clinically, cancer-induced facial pain can be evoked or occurs spontaneously and its intensity is characterized as moderate to severe (Lam & Schmidt, 2011; Viet & Schmidt, 2012). The current treatment protocols follow the analgesic ladder recommendation of the World Health Organization (WHO) for cancer pain management (Mercadante & Fulfaro, 2005; Mercadante & Giarratano, 2013; Mercadante, 2010). However, studies demonstrated that a significant proportion of patients have unsatisfactory pain control and present many side effects that markedly reduce their quality of life
(Dy et al., 2008; Foley, 2004). Thus, a better understanding of the pathophysiology of cancer-induced facial pain and the improvement of therapeutic strategies for pain control are clearly warranted (Lam & Schmidt, 2011; Schmidt, 2014, 2015). There is mounting evidence for the involvement of endothelins in many processes related to facial cancer, including pain (McKenzie, Hinsley, Hunter, & Lambert, 2014; Pickering, Jay Gupta, Quang, Jordan, & Schmidt, 2008; Quang & Schmidt, 2010b; Russo et al., 2010). Increased serum levels of endothelin-1 (ET-1) have been detected in patients with facial cancer (Pickering, Jordan, & Schmidt, 2007), as well as in tumor-bearing animals, and these alterations have been associated with tumor progression and the development of spontaneous and evoked nociception (Connelly & Schmidt, 2004; Fujita, Andoh, Saiki, & Kuraishi, 2008; Lam & Schmidt, 2011; Pickering et al., 2008;
⁎
Corresponding author at: University of Arizona, College of Medicine, Department of Pharmacology, Tucson, AZ, USA. E-mail address: [email protected] (C.M. Kopruszinski). 1 Present address: University of Arizona, College of Medicine, Department of Pharmacology, Tucson, AZ, USA. https://doi.org/10.1016/j.archoralbio.2018.10.038 Received 15 July 2018; Received in revised form 30 October 2018; Accepted 31 October 2018 0003-9969/ © 2018 Published by Elsevier Ltd.
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Aldrich, San Francisco, USA) at 30 μg/50 μL, the peptidic endothelin ETA and ETB receptor antagonists BQ-123 and BQ-788 (American Peptides, San Francisco, USA) were injected alone or in combination at 20 μg/50 μL, whereas the non-peptidic dual endothelin ETA and ETB receptor antagonist bosentan monohydrate (Actelion Pharmaceuticals, Allschwill, Switzerland) was administered at 10 or 30 μg/50 μL. Controls animals received a 50 μL injection of saline. The choice of drug doses were based on the literature (Chichorro, Zampronio, Cabrini, Franco, & Rae, 2009, 2010; Kopruszinski et al., 2018; Remeniuk et al., 2015).
Schmidt et al., 2007; Yan, Peng, & Huang, 2015). Moreover, preclinical studies in different cancer models have demonstrated the efficacy of endothelin receptor blockade on tumor-induced sensory changes. These studies highlight that the endothelin system is implicated in tumor-induced sensory changes associated with cancer cells inoculation (Fujita et al., 2008; Hamamoto, Khasabov, Cain, & Simone, 2008; Quang & Schmidt, 2010a; Schmidt et al., 2007). We recently demonstrated that systemic blockade of endothelin ETA and ETB receptors abolished tumor-induced evoked and ongoing nociception in a rat model of facial carcinoma. It was also shown that systemic treatment with an endothelin receptor antagonist potentiated morphine-induced antinociception, indicating that endothelin receptor blockade may represent an improved new treatment strategy for patients with head and neck cancer pain (Kopruszinski et al., 2018). However, the involvement of the peripheral endothelin system in evoked and ongoing nociception related to facial cancer has not yet been characterized. In light of these considerations, this study examined the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception.
2.4. Heat Stimulation
2. Material and methods
Heat sensory thresholds of the vibrissal pad area were assessed as previously reported (Chichorro et al., 2009). Briefly, each animal was lightly restrained by the experimenter while a ∼50 °C radiant heat source was positioned at 1 cm from the surface of the right vibrissal pad. The response latency to display either head withdrawal or vigorous flicking of the snout was recorded and adopted as a behavioral index of heat sensitivity. A 20 s cut-off time was used to prevent tissue damage.
2.1. Animals
2.5. Spontaneous grooming evaluation
This study was conducted on male Wistar rats weighing 180–220 g. Animals were housed in a temperature-controlled room, under 12 h dark/light circadian cycle, with free access to chow and tap water. All experimental procedures were performed in accordance with the ARRIVE guidelines, with the ethical guidelines of the International Association for the Study of Pain regulations on animal welfare and the National Institutes of Health guide for the care and use of laboratory animals. The experiments were previously approved by Federal University of Parana - Institutional Committee on the Ethical Use of Animals (authorization # 645). This study used 396 rats total, 366 animals for all the behaviors experiments and 30 animals for in vivo maintenance of the tumor cells. To determine the sample size is a requirement of the Committee on the Ethical Use of Animals of our institution before the approval of the experimental Protocols. We have determined the sample size by using the GPower 3.1 software, which, considering a significance level of 5%, estimated a minimum of 5 rats for experiments assessing thermal hyperalgesia, 11 rats for the conditioned place preference evaluation and 17 for the analysis of facial grooming. All efforts were made to diminish the number of animals used and their suffering along the study.
Evaluation of spontaneous facial grooming was performed as previously described (Kopruszinski et al., 2018). In brief, rats were submitted to individual evaluation of time spent performing spontaneous facial grooming across a 10-minutes session conducted between 8:0011:00 h. Facial grooming behavior was defined as bilateral cleaning of the face, at the vibrissae region, with the front paws (Hidaka et al., 2011). 2.6. Conditioned place preference Evaluation of ongoing/tonic nociception relief using the conditioned place preference (CPP) paradigm was performed as previously described (King et al., 2009). The CPP apparatus consist of three chambers designated as two conditioning chambers, with each compartment measuring 36 × 25 × 40 cm, with distinct visual and tactile cues, i.e. different-patterned walls (one chamber with black walls and the other one with striped walls) and texturally distinct floors (black chamber with smooth floor and stripped chamber with textured floor), respectively, separated by a neutral chamber measuring 36 × 25 × 10 cm, that was brightly lit (LED light 100 lx). The CPP paradigm consists in three steps, pre-conditioning or baseline, conditioning and test. The pre-conditioning step, defined as the first day of CPP paradigm, animals were located into the neutral chamber with free access to all chambers, and left to explore the chambers for 15 min. Exploratory behavior was filmed and recorded, with the baseline of the time-spent in each chamber analyzed. To avoid preference for one of the chambers on the baseline, rats that spent more than 80% or less than 20% in one of the conditioning chambers were removed from the study. Drug pairing was assigned to the conditioning chambers using an unbiased CPP design, with equivalent mean times across the two conditioning chambers. Drug assignment was counterbalanced across the conditioning chambers according to the amount of the time spent in each chamber during the baseline step, i,e, half of the animals were paired to the drug treatment in one of the conditioning chambers for all groups and the other half were paired to the drug treatment on the opposite conditioning chamber. A two conditioning days CPP protocol was chosen based on the drug route treatment, whereby the conditioning step occurred across two consecutive days, as detailed below. On the test day, rats were once again placed into the neutral chamber with free access to all chambers, and allowed to explore the apparatus for 15 min. Time spent in each compartment was analyzed to determine whether drug treatment produced CPP, indicated by an increased time
2.2. Maintenance and inoculation of the tumor cells To induce facial carcinoma in rats, Walker-256B lineage cells were used as previously described (Kopruszinski et al., 2018). The cells were maintained in vivo by weekly intraperitoneal (ip) passages. To induce the facial cancer, the animals were anesthetized with a solution of xylazine/ketamine (7.5/60 mg/kg, respectively, ip) and injected subcutaneously (sc) with a suspension of 2 × 106 Walker-256B cells in 100 μL into the right vibrissal pad. Control animals (sham) were similarly treated with 100 μL of vehicle (phosphate-buffered saline, PBS). The day of inoculation of the Walker-256B cells was considered day 0. From the total animals used for the behavior experiments, less than 5% did not develop tumor at the facial region. These animals were excluded of the experiments. 2.3. Drugs All drugs evaluated in the present study were diluted in 0.9% NaCl (i.e. saline), and administered locally, through sc injection into the rat’s right upper lip. Lidocaine hydrochloride (Cristália, Sao Paulo, Brazil) was administered at 1 mg/50 μL, morphine hydrochloride (Sigma 232
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in seconds spent in the drug-paired chamber as compared to pre-conditioning time and post-conditioning vehicle-paired chamber time, with positives scores indicating preference. 2.7. Behavioral analysis protocols The effects of local treatment with lidocaine, morphine (used as control drugs) or with endothelin receptor antagonists on tumor-induced heat hyperalgesia was assessed in different cohorts of rats, on day 6 post-inoculation. A total of 120 rats were used for this experiment. Baseline responsiveness to heat stimulation was evaluated solely on the right (ipsilateral) vibrissal pad prior (BL) to inoculation of tumor cells and again on day 6 (D6) to verify tumor-induced heat hyperalgesia. Following confirmation of heat hyperalgesia on day 6, bosentan, BQ123, BQ-788, BQ-123 plus BQ-788 in association, lidocaine, morphine or vehicle were all administered locally into the right upper lip. Heat hyperalgesia was re-evaluated at 30 min and again every hour up to 4 h after drug or vehicle treatment. The effect of the same treatments was assessed 30 min after their administration on tumor-induced increased spontaneous grooming in different cohorts of animals on day 6 post tumor-inoculation. A total of 213 rats were used for this experiment. In addition, CPP to local bosentan, lidocaine or morphine treatments was evaluated in separate cohorts of rats, on days 3–6 post tumor-inoculation. The pre-conditioning day occurred on day 3 after tumor cell inoculation as described above. The two conditioning days (days 4 and 5 after tumor cells inoculation) were divided in morning and afternoon sections. In the morning, rats were injected locally with vehicle and immediately confined to the appropriate pairing chamber for 30 min. Four hours later (in the afternoon), the same animals were injected locally with bosentan, lidocaine or morphine and immediately confined into the opposite pairing chamber for 30 min. The test day took place on day 6 after tumor cell inoculation. A total of 33 rats were used for this experiment. The timeline of the experimental procedures is illustrated in Fig. 1.
Fig. 1. Experimental protocols timeline. A. Heat stimulation: on day 0, baseline to facial heat stimulation was collected followed by the inoculation of tumor cells into animal’s right vibrissal pad. On day 6 after inoculation, facial heat stimulation measurement was performed to confirm the development of heat hyperalgesia. Treatment was performed subcutaneously (right upper lip) and facial heat stimulation was assed hourly. B. Spontaneous facial grooming: on day 0, animals were submitted to the inoculation of tumor cells into animal’s right vibrissal pad. On day 6 after inoculation, treatment was performed subcutaneously (right upper lip) and spontaneous facial grooming was assessed for 10 min after the treatment. C. Conditioned place preference (CPP): on day 0, animals were submitted to the inoculation of tumor cells into animal’s right vibrissae pad. On day 3, drug-free animals were placed into CPP apparatus with free access to all three chambers for baseline collection. On days 4 and 5, rats were exposed to two conditionings days. In the mornings (AM), animals were treated with vehicle and conditioned to a designed chamber, with no access to the other chambers. In the afternoons (PM), animals were treated with drug and conditioned to the opposite chamber to the morning section, also with no access to the other chambers. On day 6, drug-free animals were submitted to the test day, with free access to all three chambers, for the evaluation of conditioned place preference.
2.8. Statistical analysis Data are presented as mean ± S.E.M. and n represent the number of rats analyzed. Heat hyperalgesia time-course data for drug effects were analyzed using two-way ANOVA followed by Tukey post-hoc tests. For spontaneous grooming and CPP data, the effects of treatment were analyzed by two-way ANOVA followed by Tukey post-hoc test between baseline (pre-conditioning) and test (post-conditioning) values. All data were analyzed with GraphPad Prism® 6. A statistically difference was set to a probability level of 0.05. Error bars are S.E.M. 3. Results 3.1. Effect of local endothelin receptor blockade on tumor-induced heat hyperalgesia
peptidic dual ETA/ETB endothelin receptor antagonist bosentan attenuated the tumor-induced heat hyperalgesia for one hour, starting within 30 min of treatment, at both doses tested (Fig. 2C, p < 0.05 versus sham rats treated with vehicle and p < 0.05 versus tumorbearing rats treated with vehicle). Sham animals treated with vehicle or bosentan did not display any difference in response latencies at any time point (Fig. 2C, p > 0.05).
Tumor-bearing rats developed heat hyperalgesia (i.e. a decrease in response latency relative to baseline), which was attenuated by local control treatments, lidocaine and morphine. Both treatments significantly reduced tumor-induced heat hyperalgesia within 30 min of treatment, and the response latencies returned to pre-lidocaine and premorphine values by 1 and 2 h post-administration, respectively (Fig. 2A, p < 0.05 versus sham rats treated with vehicle and p < 0.05 verustumor-bearing rats treated with vehicle). Sham animals treated with lidocaine or morphine did not show any difference in response latencies when compared to vehicle-treated controls at any time-point (Fig. 2A, p > 0.05). On day 6 post inoculation, local treatment with BQ-123 or BQ-788, either alone or in combination, failed to modify the heat hyperalgesia induced by tumor cell inoculation (Fig. 2B, p < 0.05 versus sham rats treated with vehicle). In contrast, similar treatment with the non-
3.2. Effects of local endothelin receptor blockade on tumor-induced increase in facial grooming On day 6 post tumor cell inoculation, tumor-bearing rats displayed increased spontaneous grooming relative to sham controls (Fig. 3A, p < 0.01 versus sham rats treated with vehicle). Local administration of the control drugs, lidocaine or morphine blocked the increase in spontaneous grooming of tumor bearing rats to values equivalent to those shown by sham controls (Fig. 3A, p < 0.05 versus tumor-bearing 233
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Fig. 2. Effect of local administration of lidocaine, morphine and endothelin receptor antagonists on tumor-induced facial heat hyperalgesia. A. Local treatment with lidocaine and morphine reduced heat hyperalgesia in tumorbearing rats on day 6 post inoculation by 30 min after administration (*p < 0.05 versus Sham-Vehicle and #p < 0.05 versus Tumor-Vehicle, n=58). B. Local administration of BQ-123 and BQ-788, either alone or in combination, failed to modify tumor-induced heat hyperalgesia (*p < 0.05 versus Sham-Vehicle, n=6-10). C. Local treatment with bosentan, at both doses, reduced heat hyperalgesia in tumor-bearing rats by 30 min post administration (*p < 0.05 versus Sham-Vehicle and #p < 0.05 versus Tumor-Vehicle, n=612). Results are presented as mean ± S.E.M. and statistical analysis was assessed by two-way ANOVA followed by Tukey test.
modify the tumor-induced increase in grooming behavior when tested at the lower dose of 10 μg (Fig. 3C, p > 0.05versus tumor-bearing rats treated with vehicle). Nonetheless, local bosentan treatment at the higher dose of 30 μg effectively blocked tumor-induced increase in grooming, with the time spent grooming equivalent to that of sham controls (Fig. 3C, p < 0.001 versus tumor-bearing rats treated with vehicle and p > 0.05 versus sham rats treated with vehicle). Also, bosentan at 30 μg did not modify the grooming behavior of sham rats (Fig. 3C, p > 0.05versus sham rats treated with vehicle). 3.3. Local lidocaine, morphine and bosentan treatment failed to control ongoing nociception related to facial cancer Local treatments with bosentan (30 μg/50 μl), lidocaine (1 mg/ 50 μl) or morphine (30 μg/50 μL) failed to induce CPP on the test day (i.e. day 6 after tumor cell inoculation), as evidenced by the absence of any significant alterations in the time spent in the pairing chambers following two conditioning sessions on days 4 and 5 after inoculation. Pre-conditioning (baseline) scores measured on day 3 after inoculation did not differ between the vehicle-paired chamber and the drug-paired chamber. On the test day, local lidocaine, morphine and bosentan treatments did not increase time spent in the drug-paired chamber of tumor-bearing animals when compared to pre-conditioning scores measures (Table 1). 4. Discussion This study showed the ability of peripheral blockade of both ETA and ETB endothelin receptors in controlling the facial heat hyperalgesia and increased facial grooming behavior in a model of facial cancer in rats. On the other hand, the current data demonstrate that peripheral dual blockade of both ETA and ETB receptors failed to modify tumorinduced ongoing facial nociception. These data suggest that peripheral ETA and ETB receptors mediate responses related to evoked nociception and increased grooming induced by facial cancer. We recently demonstrated that systemic treatment with the dual endothelin ETA and ETB receptor antagonist bosentan abolished tumorinduced heat hyperalgesia, increased spontaneous grooming and ongoing nociception in the same facial cancer model used in the present study (Kopruszinski et al., 2018). However, the contribution of peripheral endothelins was not investigated. Herein, it was shown that local treatment with bosentan blocked facial heat hyperalgesia and increased facial grooming in tumor-bearing rats. This effectiveness was similar to that promoted by local treatment with morphine and lidocaine, used as control drugs in this study. On the other hand, local treatments with the peptidic endothelin receptors antagonists BQ-123 and BQ-788, alone or in association, were unable to reduce the facial heat hyperalgesia or increased facial grooming of tumor-bearing rats. This ineffectiveness might be a result of increased synthesis and release of proteases by the tumor cells, resulting in the hydrolysis, and consequently, the degradation of the BQ molecules prior to receptor binding (Hardt, Lam, Dolan, & Schmidt, 2011; Schmidt, 2014). Thus, these data suggest that local both receptors contribute to the development of facial
rats treated with vehicle). Local administration of lidocaine or morphine to sham rats failed to alter grooming behavior (Fig. 3A, p > 0.05). Local administration of BQ-123 or BQ-788, either alone or in association, failed to reduce the increase in grooming behavior observed on day 6 after tumor cell inoculation (Fig. 3B, p < 0.01, p < 0.05 and p < 0.05 versus Sham-Vehicle). Likewise, bosentan also failed to 234
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Table 1 Effect of local administration of lidocaine, morphine or dual ETA/ETB endothelin receptor antagonist bosentan on tumor-induced ongoing/tonic nociception. Local treatment with lidocaine, morphine or bosentan failed to induce CPP on the test day (day 6 post tumor cell inoculation), as attested by absence of significant increases in the time spent in seconds in the drug-paired chamber in a CPP paradigm, when compared to pre-conditioning (baseline) scores measures (p > 0.05 versus BL, n = 10–12). Results are presented as mean ± SEM and statistical analysis was assessed by two-way ANOVA followed by Tukey test. Time spent in drug-paired chamber (seconds) Group
Baseline
Test
Lidocaine Morphine Bosentan
396.1 ± 40.5 403.0 ± 28 396.9 ± 29.4
453.2 ± 89.4 466.5 ± 94.8 349.8 ± 77.5
heat hyperalgesia associated to facial cancer and that dual endothelin receptor blockade with a non-peptidic drug was effective to control tumor-induced heat hyperalgesia. The contribution of local ETA and ETB receptors towards heat hyperalgesia induced by inoculation of XC tumor cells 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 BQ123 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). In addition to these hypotheses, other factors related to the pharmacokinetics of BQs and bosentan, such as their distribution profile, or related to the receptor binding affinity may account to the difference in effectiveness, but these aspects remain to be investigated. 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,
Fig. 3. Effect of local administration of lidocaine, morphine and endothelin receptor antagonists on tumor-induced facial grooming. A. Tumor inoculation increased facial grooming behavior on day 6 post inoculation. Local treatment with lidocaine or morphine reduced the facial grooming in tumor-bearing rats (**p < 0.01 versus Sham-Vehicle and #p < 0.05 versus Tumor-Vehicle, n=11-14). B. Local administration of BQ-123 and BQ-788 alone or associated failed to modify tumor-induced facial grooming (*p < 0.05 and **p < 0.01 versus Sham-Vehicle, n=8-18). C. Local treatment with bosentan reduced the facial grooming in tumor-bearing rats only at the highest dose (***p < 0.001 versus Sham-Vehicle and ###p < 0.001 versus Tumor-Vehicle, n=11-22). Results are presented as mean ± S.E.M. and statistical analysis was assessed by two-way ANOVA followed by Tukey test.
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of Higher Education Personnel (CAPES) during the study development.
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 tumorbearing 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). Altogether, the results of this study demonstrate that peripheral blockade of both ETA and ETB receptors reduces tumor-induced facial heat hyperalgesia and increased spontaneous grooming, without modifying ongoing nociception. It is possible to suggest that bosentan is acting upon both receptors present in primary sensory afferents, or is causing the blockade of ETA, and mainly ETB receptors, present in tumor cells, promoting the blockade of endothelin-1 release from these cells, which in both cases will prevent peripheral nociceptor sensitization (Barr, Kam, Khodorova, Montmayeur, & Strichartz, 2011; Khodorova et al., 2009; Schmidt, Hamamoto, Simone, & Wilcox, 2010; Schmidt, 2014; Smith et al., 2014). The current findings contribute to the knowledge of the pathophysiology of cancer-induced facial pain and may facilitate the clinical exploration of the benefits of endothelin receptor blockade as a useful adjuvant strategy for facial cancer pain management.
Conflict of interest The authors declare no conflicts of interest. Acknowledgements The group is grateful to CNPq and CAPES for their funding support. We thank Dr. Sandra Coccuzzo Sampaio (Butantan Institute, Sao Paulo, Brazil) for kindly donating the Walker-256B tumor cells. We also thank Dr. Alexandra Acco, M.Sc. Eder Gambeta and M.Sc. Larissa Galuppo for technical support. References Akiyama, T., Carstens, M. I., & Carstens, E. (2010). Spontaneous itch in the absence of hyperalgesia in a mouse hindpaw dry skin model. Neuroscience Letters, 484(1), 62–65. https://doi.org/10.1016/j.neulet.2010.08.020. Awano, S., Dawson, L. A., Hunter, A. R., Turner, A. J., & Usmani, B. A. (2006). Endothelin system in oral squamous carcinoma cells: Specific siRNA targeting of ECE-1 blocks cell proliferation. International Journal of Cancer, 118(7), 1645–1652. https://doi. org/10.1002/ijc.21525. Baamonde, A., Lastra, A., Fresno, M. F., Llames, S., Meana, A., Hidalgo, A., ... Menendez, L. (2004). 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Funding sources This study was supported by National Council for Scientific and Technological Development (CNPq), grant #477679/2012-9. The first author received PhD. scholarship by Coordination for the Improvement 236
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