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Evaluation of heat hyperalgesia and anxiety like-behaviors in a rat model of orofacial cancer
Neuroscience Letters 619 (2016) 100–105
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Research paper
Evaluation of heat hyperalgesia and anxiety like-behaviors in a rat model of orofacial cancer Eder Gambeta, Caroline Machado Kopruszinski, Renata Cristiane dos Reis, Janaina Menezes Zanoveli ∗ , Juliana Geremias Chichorro Department of Pharmacology, Biological Sciences Building, Federal University of Parana, Curitiba, PR, Brazil
h i g h l i g h t s • • • •
Rats with orofacial cancer developed heat hyperalgesia. Anxiety-like behavior was observed in rats with orofacial cancer. Lidocaine showed a transient anti-hyperalgesic effect, but did not affect anxiety-like behaviors. Midazolam reduced the anxiety-like behaviors, but did not modify heat hyperalgesia.
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Article history: Received 21 January 2016 Received in revised form 29 February 2016 Accepted 1 March 2016 Available online 4 March 2016 Keywords: Hyperalgesia Anxiety Cancer Lidocaine Midazolam Rats
a b s t r a c t Pain and anxiety are commonly experienced by cancer patients and both significantly impair their quality of life. Some authors claim that there is a relationship between pain and anxiety, while others suggest that there is not a direct association. In any case, there is indeed a consensus that anxiety impairs the pain condition beyond be under diagnosed and undertreated in cancer pain patients. Herein we investigated if rats presenting heat hyperalgesia induced by orofacial cancer cell inoculation would display anxiety-like behaviors. In addition, we evaluated if pain blockade would result in alleviation of anxiety behaviors, as well as, if blockade of anxiety would result in pain relief. Orofacial cancer was induced in male Wistar rats by inoculation of Walker-256 cells into the right vibrissal pad. Heat facial hyperalgesia was assessed on day 6 after the inoculation, and on this time point rats were submitted to the elevated plus maze and the light-dark transition tests. The influence of lidocaine and midazolam on heat hyperalgesia and anxietylike behaviors was assessed. The peak of facial heat hyperalgesia was detected 6 days after cancer cells inoculation, and at this time point, rats exhibited increased anxiety-like behaviors. Local treatment with lidocaine (2%/50 L) caused a marked reduction of heat hyperalgesia, but failed to affect the anxiety-like behaviors, while midazolam (0.5 mg/kg, i.p.) treatment failed to change the heat threshold, but induced an anxiolytic-like effect. Altogether, our data demonstrated that rats with orofacial cancer present painand anxiety-like behaviors, but brief heat hyperalgesia relief does not affect the anxiety-like behaviors, and vice-versa, in our experimental conditions. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Head and neck cancer (HNC) encompasses a heterogeneous group of tumors originating from the tissues and organs of the head and neck that together comprise the seventh most frequent cancer worldwide [1,2]. Pain is commonly associated with HNC, as 85% of the patients report oral pain at the time of diagnosis [3,4].
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J.M. Zanoveli). http://dx.doi.org/10.1016/j.neulet.2016.03.001 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
The etiology of orofacial pain is multifactorial and may be related with the disease and/or the treatment. Cancer pain during and following the treatment has been correlated with increased morbidity, impaired performance status, increased anxiety and depression with a reduction in the quality of life [5]. Of particular interest, there is evidence that anxiety plays an important role in modulating pain experience in cancer patients [6]. While some authors believe that there is a relationship between physical symptoms and the presence of anxiety, others did not find a correlation between these factors. In any case, it is widely accepted in the clinical setting that the anxiety is under
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recognized and consequently undertreated in cancer patients receiving palliative care [7–10]. In spite of the accumulating evidence that pain and anxiety are commonly experienced by cancer patients, few studies have evaluated both factors in HNC patients and the relationship between these factors have not been well studied. Consequently, the aim of this study was to investigate the presence of anxiety-like behaviors in rats presenting heat orofacial hyperalgesia induced by inoculation of Walker 256B tumor cells into the vibrissal pad. In order to explore a possible correlation between pain and anxiety we evaluated if pain blockade would result in alleviation of anxiety-like behaviors, as well as, if the blockade of anxiety would result in pain relief in rats with orofacial cancer.
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of entries in each arm (open and enclosed) was evaluated. If the animal falls out of the open arm during the test, it was excluded. 2.5. Light-dark transition (LDT) test The test was conducted according to Vicente and Zangrossi [16]. The animal was placed in the middle of the lit compartment facing the doorway separating the two compartments. After the first transition to the dark compartment, the behavior of the animal was recorded for an additional 5 min period. During this period, the total time spent in the lit compartment and the number of transitions between the two compartments was registered. 2.6. Drugs
2. Materials and methods 2.1. Animals Experiments were conducted on male Wistar rats weighing 180–220 g, maintained five animals per cage at controlled temperature (22 ± 1 ◦ C) under 12/12 h light/dark cycle (lights on at 07:00 h) with chow and water ad libitum. They were acclimatized in the laboratory for at least 48 h before use. Experimental procedures were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain [11] and approved by UFPR’s institutional Committee on the Ethical Use of Animals (authorization #848) and all efforts were made to minimize the number of animals used and their suffering. 2.2. Maintenance and inoculation of the tumor cells Walker 256B-cells were used to induce orofacial cancer in rats, as previously described [12], with minor modifications. The cells were obtained by inoculating 1 × 107 (1 mL) tumor cells into the peritoneal cavity of the rats. Their maintenance was carried out by weekly passages through intraperitoneal inoculation at the same concentration. After 5–7 days, animals were euthanized and their ascitic fluids were collected in a solution of ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8.0, 1:1). The viability of tumor cells was assessed by the Trypan blue exclusion method [13]. To induce the facial cancer, the animals were anesthetized intraperitoneally with xilazine (7.5 mg/kg) and ketamine (60 mg/kg) solution and 2 × 106 cells/100 L were injected subcutaneously into the right vibrissal pad. Control animals received the same volume of vehicle (phosphate-buffered saline, PBS). 2.3. Heat stimulation Thermal hyperalgesia on the orofacial area was measured as previously described [14]. Briefly, the animal was gently held by the experimenter and a radiant heat source was presented 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 (in s) using a stopwatch, and to prevent tissue damage, a 20 s cut-off time was stablished. Reaction to heat stimulation was assessed before (basal responsiveness) and on day 6 after inoculation of the cells or its vehicle. 2.4. Elevated plus-maze (EPM) test The test was carried out as previously described [15] on day 6 after tumor inoculation, by placing the animal in the center of the apparatus followed by record of its behavior for 5 min. The time that each animal remained in the open arms, as well as the number
Lidocaine and midazolam were obtained from Cristália Produtos Químicos Farmacêuticos (Itapira, SP, Brazil) and Hipolabor Farmacêutica (Sabará, MG, Brazil), respectively, and both were dissolved in sterile saline solution. The doses of lidocaine and midazolam were based on previous studies [17,18] and in a pilot study conducted in our laboratory (data not shown). Ketamine was obtained from Rhobifarma Ind. Farmacêutica (Hortolândia, SP, Brazil) and xylazine from Laboratórios König S.A. (Avellaneda, Argentina). 2.7. Experimental protocols The response latency to facial heat stimulation was assessed before (pre) and 6 days after tumor cells inoculation, followed by rats treatment with a single injection of lidocaine (2%, 50 L, s.c), midazolam (0.5 mg/kg, i.p.) or its corresponding vehicles. Heat hyperalgesia was evaluated at 30 and 15 min-intervals, respectively, up to 2 h after the treatments. In an independent group, 6 days after inoculation of tumor cells, rats were treated with lidocaine (2%, 50 L, s.c), midazolam (0.5 mg/kg, i.p.) or its corresponding vehicles and 30 and 15 min after, respectively, the animals were submitted to the EPM and LDT tests. 2.8. Statistical analysis All data are presented as mean ± S.E.M. (standard error of the mean). Two-way ANOVA with or without repeated measures followed by the Bonferroni post-hoc test was used to analyze the data, with condition (tumor or control) and/or drug treatment and/or time as the independent factors. When only condition factor was used as independent factor, Student t-test was applied. Results were considered statistically significant if p < 0.05. 3. Results 3.1. Local lidocaine treatment reduced heat hyperalgesia but failed to modify anxiogenic-like behaviors in tumor bearing rats Tumor cells inoculation at the orofacial region induced the development of facial heat hyperalgesia, which was significantly reduced by local treatment with lidocaine (2%/50 L) at 30 min after the treatment, compared with the control group (Fig. 1A, p < 0.05). Lidocaine treatment did not modify the response latency to the heat stimulus of animals inoculated with vehicle (Fig. 1A, p > 0.05). Tumor-bearing rats demonstrated an anxiogenic-like behavior (time spent on the lit compartment on LDT [condition factor: F = (1,22) = 15.50; p < 0.05], (Fig. 1B) and time spent in the open arm of EPM [condition factor: F = (1,22) = 23.18; p < 0.05]), (Fig. 1C) at the same point as heat hyperalgesia was detected (i.e. 6 days after cells inoculation). Local treatment with lidocaine failed to induce an anxiolytic-like effect evaluated 30 min after its injection (time in the lit compartment of LDT test [treatment
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Fig. 1. Effect of local lidocaine treatment in animals submitted to the heat hyperalgesia, elevated plus maze (EPM) and light-dark transition (LDT) tests after tumor cells inoculation. (A) Heat hyperalgesia time course. (B) Percentage of time spent in the lit compartment of the LDT test. (C) Percentage of time spent in the open arms of the EPM test. (D) Percentage of entries in the open arms of the EPM test. * and # p < 0.05 vs. Veh–Veh and Tumor-Veh, respectively (Fig. A, n = 7–9); * p < 0.05 when compared to Veh–Veh (B, C, n = 6–8). All data are expressed as mean ± S.E.M.
factor: F(1,22) = 0,0006397; p > 0.05]), (Fig. 1B), time spent in the open arms of EPM test [treatment factor: F(1,22) = 1.881; p > 0.05], (Fig. 1C). Moreover, it was not observed statistical difference in the open arm entries [condition factor: F(1,22) = 2.498; p > 0.05; treatment factor: F(1,22) = 0,1045; p > 0.05], (Fig. 1D). It is also important to point out that the findings are not related to a locomotor deficit, since no changes in the number of transitions evaluated in the LDT or in the number of entries into the enclosed arms of the EPM were detected (data not shown). 3.2. Systemic midazolam treatment reduced anxiogenic-like behaviors but failed to attenuate thermal hyperalgesia after inoculation of tumor cells Inoculation of tumor cells caused a significant orofacial heat hyperalgesia on day 6 after the procedure (Fig. 2A, p < 0.05) which was not affected by systemic treatment with midazolam (0.5 mg/kg, Fig. 2A, p > 0.05). In an independent group of animals it was observed an anxiogenic-like behavior in tumor-bearing rats (time in the lit compartment of LDT test [condition factor: F(1,35) = 17.41; p < 0.05], Fig. 2B). Systemic treatment with midazolam was able to induce an anxiolytic-like effect both in the control and tumor-bearing rats assessed 15 min after the treatment [treatment factor: F(1,35) = 9.093, p < 0.05], (Fig. 2B). Despite the statistical analysis do not show effect on the condition factor [F(1,34) = 0,7359; p = 0.397], it was clearly observed an effect on the treatment factor [F(1,34) = 72.83, p < 0.05] and also the interaction between the factors treatment and condition [interaction factor: F(1,34) = 8.976; p < 0.05]. Also, no statistically significant difference was observed on open arm entries [condition factor: F(1,34) = 0,07114; p > 0.05; treatment factor: F(1,34) = 3.382; p > 0.05], (Fig. 1D).
4. Discussion The present data showed that tumor-bearing rats present an anxiogenic-like behavior, in addition to the nociceptive behavior. Local treatment with lidocaine reduced the heat hyperalgesia, without changing the anxiety-like behaviors, while midazolam treatment failed to change the heat hyperalgesia, but induced an anxiolytic-like effect in rats. The facial cancer model used in the present study has been well characterized by our group [19,unpublished observations] and others [20–22]. According to these studies, rats submitted to inoculation of Walker-256B cells into the vibrissa pad display increased spontaneous grooming, thermal and mechanical orofacial hypersensitivity starting as soon as 4 days after cells inoculation and persisting around 10 days. The nociceptive behaviors on this model are not significantly mediated by cancer-induced peripheral inflammation, suggesting the contribution of a neuropathic pain component [21]. Thus, it represents a valuable model in the study of pain mechanisms related to orofacial cancer, as well as in the study of other complications related to this condition. It has been reported that anxiety is a frequent symptom experienced by cancer patients and it is usually under diagnosed and undertreated. Moreover, limited research has been undertaken to understand its mechanism, to establish its appropriate treatment and its relation with pain [23,24]. In the current study we demonstrated that rats with orofacial cancer display nociceptive- and anxiety-like behaviors, which corroborates clinical studies that addressed the major symptoms experienced by cancer pain patients. Additionally, this finding is in agreement with some pre-clinical studies that evaluate nociceptive- and anxietylike behaviors in models of inflammatory and neuropathic pain [18,25–28]. Some of these studies reported that centrally-acting
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Fig. 2. Effect of systemic midazolam treatment in animals submitted to the heat hyperalgesia, elevated plus maze (EPM) and light–dark transition (LDT) tests after tumor cells inoculation. (A) Heat hyperalgesia time course. (B) Percentage of time spent in the lit compartment of the LDT test. (C) Percentage of time spent in the open arms of the EPM test. (D) Percentage of entries in the open arms of the EPM test. * and # p < 0.05 vs. Veh–Veh and Tumor-Veh, respectively, (A, n = 6–10); * p < 0.05 vs. Veh–Veh and # p < 0.05 vs. its respective control group (B, C, n = 8–15). All data are expressed as mean ± S.E.M.
analgesics, such as morphine, tramadol and gabapentin, are able to relieve anxiety-like behaviors in chronic pain models probably via their anti-nociceptive properties [18,29]. Likewise, recent studies have suggested that there are common central sites that play a role in the regulation of pain sensation and anxiety-like behaviors [30,31]. Thus, according to the current literature, anxiolytic and analgesic effects can be simultaneously achieved by targeting specific central sites, such as neurons in the anterior cingulate cortex or in the prelimbic cortex [30,31]. In line with this observation, we demonstrated that peripheral nerve block with lidocaine was able to attenuate heat hyperalgesia in tumor-bearing rats, but did not influence the anxiety-like behaviors. We do not believe that this result is specific to the sensorial modality assessed herein (i.e. heat hyperalgesia), since unpublished observations of our group demonstrated a positive correlation of anxiety-like behaviors and mechanical allodynia in tumor-bearing rats. In addition, we suggest that the lack of effect of lidocaine in anxiety-like behaviors is mainly due to its restriction in the periphery, but it can be also due to the short antihyperalgesic effect promoted by lidocaine. It can be speculated that a longer period of peripheral pain relief would result in attenuation of anxiety-like behaviors. In chronic pain, nociceptors trigger an increased rate of action potentials and subsequently convey pain signals to several brain areas that are thought to be involved in the initiation of pain perception and anxiety [30–32]. Thus, it remains to be investigated if prolonged peripheral pain relief would contribute to diminish the anxiety-related parameters. Several studies have reported that anxiety can affect the perception of pain and enhance pain sensation [33–36]. Benzodiazepines are extensively indicated to treat some types of anxiety disorders, but side effects and dependence limit their long-term use [37,38]. There is no consistent clinical evidence that benzodiazepines can produce analgesic effect, but they are prescribed to chronic pain
patients to relieve anxiety [39,40]. On the other hand, few preclinical studies that have addressed the effect of benzodiazepines on pain models have generated controversial data, which can be related to the dose employed, route and period of administration, as well as pain model [41–43]. According to our data, a single injection of midazolam resulted in a marked anxiolytic-like effect, but failed to modify the heat hyperalgesia in tumor-bearing rats, which is in line with previous observations [44,45]. Indeed, this finding completely corroborates the study by Roeska and colleagues [18], which demonstrated anxiolytic-like, but not anti-hyperalgesic-like, effects of midazolam in rats submitted to chronic constriction injury of the sciatic nerve. One of the main mechanisms related to midazolam-anxiolytic effect is the enhancement of GABAA -mediated inhibition in brain areas such as amygdala, prefrontal cortex, hypothalamus and hippocampus, among others [46–48]. It is interesting to note that alterations in hippocampus neurons have been reported in models of chronic pain, but they have been related to the anxiety-like, but not nociceptive-like, behaviors presented by the animals [26,31]. Thus, it could be suggested that the lack of effect of midazolam on hyperalgesia is related to its action on a brain substrate much more related to anxiety than to pain. In addition, there is evidence that anxiety is a consequence of persistent pain, as morphine and gabapentin reversed anxiety-like behaviors in neuropathic animals without having an effect on anxiety in sham-operated rats [18]. This later study corroborates previous reports showing that pain alleviation reduced the behavioral signs of anxiety [49,50]. In conclusion, our results demonstrated that facial tumorbearing rats display nociceptive and anxiogenic-like behaviors, which are not concomitantly attenuated by brief heat hyperalgesia relief or by decreasing anxiety. However, we can not discard the possibility that these conditions are correlated, as only the
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Research paper
Evaluation of heat hyperalgesia and anxiety like-behaviors in a rat model of orofacial cancer Eder Gambeta, Caroline Machado Kopruszinski, Renata Cristiane dos Reis, Janaina Menezes Zanoveli ∗ , Juliana Geremias Chichorro Department of Pharmacology, Biological Sciences Building, Federal University of Parana, Curitiba, PR, Brazil
h i g h l i g h t s • • • •
Rats with orofacial cancer developed heat hyperalgesia. Anxiety-like behavior was observed in rats with orofacial cancer. Lidocaine showed a transient anti-hyperalgesic effect, but did not affect anxiety-like behaviors. Midazolam reduced the anxiety-like behaviors, but did not modify heat hyperalgesia.
a r t i c l e
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Article history: Received 21 January 2016 Received in revised form 29 February 2016 Accepted 1 March 2016 Available online 4 March 2016 Keywords: Hyperalgesia Anxiety Cancer Lidocaine Midazolam Rats
a b s t r a c t Pain and anxiety are commonly experienced by cancer patients and both significantly impair their quality of life. Some authors claim that there is a relationship between pain and anxiety, while others suggest that there is not a direct association. In any case, there is indeed a consensus that anxiety impairs the pain condition beyond be under diagnosed and undertreated in cancer pain patients. Herein we investigated if rats presenting heat hyperalgesia induced by orofacial cancer cell inoculation would display anxiety-like behaviors. In addition, we evaluated if pain blockade would result in alleviation of anxiety behaviors, as well as, if blockade of anxiety would result in pain relief. Orofacial cancer was induced in male Wistar rats by inoculation of Walker-256 cells into the right vibrissal pad. Heat facial hyperalgesia was assessed on day 6 after the inoculation, and on this time point rats were submitted to the elevated plus maze and the light-dark transition tests. The influence of lidocaine and midazolam on heat hyperalgesia and anxietylike behaviors was assessed. The peak of facial heat hyperalgesia was detected 6 days after cancer cells inoculation, and at this time point, rats exhibited increased anxiety-like behaviors. Local treatment with lidocaine (2%/50 L) caused a marked reduction of heat hyperalgesia, but failed to affect the anxiety-like behaviors, while midazolam (0.5 mg/kg, i.p.) treatment failed to change the heat threshold, but induced an anxiolytic-like effect. Altogether, our data demonstrated that rats with orofacial cancer present painand anxiety-like behaviors, but brief heat hyperalgesia relief does not affect the anxiety-like behaviors, and vice-versa, in our experimental conditions. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Head and neck cancer (HNC) encompasses a heterogeneous group of tumors originating from the tissues and organs of the head and neck that together comprise the seventh most frequent cancer worldwide [1,2]. Pain is commonly associated with HNC, as 85% of the patients report oral pain at the time of diagnosis [3,4].
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J.M. Zanoveli). http://dx.doi.org/10.1016/j.neulet.2016.03.001 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
The etiology of orofacial pain is multifactorial and may be related with the disease and/or the treatment. Cancer pain during and following the treatment has been correlated with increased morbidity, impaired performance status, increased anxiety and depression with a reduction in the quality of life [5]. Of particular interest, there is evidence that anxiety plays an important role in modulating pain experience in cancer patients [6]. While some authors believe that there is a relationship between physical symptoms and the presence of anxiety, others did not find a correlation between these factors. In any case, it is widely accepted in the clinical setting that the anxiety is under
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recognized and consequently undertreated in cancer patients receiving palliative care [7–10]. In spite of the accumulating evidence that pain and anxiety are commonly experienced by cancer patients, few studies have evaluated both factors in HNC patients and the relationship between these factors have not been well studied. Consequently, the aim of this study was to investigate the presence of anxiety-like behaviors in rats presenting heat orofacial hyperalgesia induced by inoculation of Walker 256B tumor cells into the vibrissal pad. In order to explore a possible correlation between pain and anxiety we evaluated if pain blockade would result in alleviation of anxiety-like behaviors, as well as, if the blockade of anxiety would result in pain relief in rats with orofacial cancer.
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of entries in each arm (open and enclosed) was evaluated. If the animal falls out of the open arm during the test, it was excluded. 2.5. Light-dark transition (LDT) test The test was conducted according to Vicente and Zangrossi [16]. The animal was placed in the middle of the lit compartment facing the doorway separating the two compartments. After the first transition to the dark compartment, the behavior of the animal was recorded for an additional 5 min period. During this period, the total time spent in the lit compartment and the number of transitions between the two compartments was registered. 2.6. Drugs
2. Materials and methods 2.1. Animals Experiments were conducted on male Wistar rats weighing 180–220 g, maintained five animals per cage at controlled temperature (22 ± 1 ◦ C) under 12/12 h light/dark cycle (lights on at 07:00 h) with chow and water ad libitum. They were acclimatized in the laboratory for at least 48 h before use. Experimental procedures were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain [11] and approved by UFPR’s institutional Committee on the Ethical Use of Animals (authorization #848) and all efforts were made to minimize the number of animals used and their suffering. 2.2. Maintenance and inoculation of the tumor cells Walker 256B-cells were used to induce orofacial cancer in rats, as previously described [12], with minor modifications. The cells were obtained by inoculating 1 × 107 (1 mL) tumor cells into the peritoneal cavity of the rats. Their maintenance was carried out by weekly passages through intraperitoneal inoculation at the same concentration. After 5–7 days, animals were euthanized and their ascitic fluids were collected in a solution of ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8.0, 1:1). The viability of tumor cells was assessed by the Trypan blue exclusion method [13]. To induce the facial cancer, the animals were anesthetized intraperitoneally with xilazine (7.5 mg/kg) and ketamine (60 mg/kg) solution and 2 × 106 cells/100 L were injected subcutaneously into the right vibrissal pad. Control animals received the same volume of vehicle (phosphate-buffered saline, PBS). 2.3. Heat stimulation Thermal hyperalgesia on the orofacial area was measured as previously described [14]. Briefly, the animal was gently held by the experimenter and a radiant heat source was presented 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 (in s) using a stopwatch, and to prevent tissue damage, a 20 s cut-off time was stablished. Reaction to heat stimulation was assessed before (basal responsiveness) and on day 6 after inoculation of the cells or its vehicle. 2.4. Elevated plus-maze (EPM) test The test was carried out as previously described [15] on day 6 after tumor inoculation, by placing the animal in the center of the apparatus followed by record of its behavior for 5 min. The time that each animal remained in the open arms, as well as the number
Lidocaine and midazolam were obtained from Cristália Produtos Químicos Farmacêuticos (Itapira, SP, Brazil) and Hipolabor Farmacêutica (Sabará, MG, Brazil), respectively, and both were dissolved in sterile saline solution. The doses of lidocaine and midazolam were based on previous studies [17,18] and in a pilot study conducted in our laboratory (data not shown). Ketamine was obtained from Rhobifarma Ind. Farmacêutica (Hortolândia, SP, Brazil) and xylazine from Laboratórios König S.A. (Avellaneda, Argentina). 2.7. Experimental protocols The response latency to facial heat stimulation was assessed before (pre) and 6 days after tumor cells inoculation, followed by rats treatment with a single injection of lidocaine (2%, 50 L, s.c), midazolam (0.5 mg/kg, i.p.) or its corresponding vehicles. Heat hyperalgesia was evaluated at 30 and 15 min-intervals, respectively, up to 2 h after the treatments. In an independent group, 6 days after inoculation of tumor cells, rats were treated with lidocaine (2%, 50 L, s.c), midazolam (0.5 mg/kg, i.p.) or its corresponding vehicles and 30 and 15 min after, respectively, the animals were submitted to the EPM and LDT tests. 2.8. Statistical analysis All data are presented as mean ± S.E.M. (standard error of the mean). Two-way ANOVA with or without repeated measures followed by the Bonferroni post-hoc test was used to analyze the data, with condition (tumor or control) and/or drug treatment and/or time as the independent factors. When only condition factor was used as independent factor, Student t-test was applied. Results were considered statistically significant if p < 0.05. 3. Results 3.1. Local lidocaine treatment reduced heat hyperalgesia but failed to modify anxiogenic-like behaviors in tumor bearing rats Tumor cells inoculation at the orofacial region induced the development of facial heat hyperalgesia, which was significantly reduced by local treatment with lidocaine (2%/50 L) at 30 min after the treatment, compared with the control group (Fig. 1A, p < 0.05). Lidocaine treatment did not modify the response latency to the heat stimulus of animals inoculated with vehicle (Fig. 1A, p > 0.05). Tumor-bearing rats demonstrated an anxiogenic-like behavior (time spent on the lit compartment on LDT [condition factor: F = (1,22) = 15.50; p < 0.05], (Fig. 1B) and time spent in the open arm of EPM [condition factor: F = (1,22) = 23.18; p < 0.05]), (Fig. 1C) at the same point as heat hyperalgesia was detected (i.e. 6 days after cells inoculation). Local treatment with lidocaine failed to induce an anxiolytic-like effect evaluated 30 min after its injection (time in the lit compartment of LDT test [treatment
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Fig. 1. Effect of local lidocaine treatment in animals submitted to the heat hyperalgesia, elevated plus maze (EPM) and light-dark transition (LDT) tests after tumor cells inoculation. (A) Heat hyperalgesia time course. (B) Percentage of time spent in the lit compartment of the LDT test. (C) Percentage of time spent in the open arms of the EPM test. (D) Percentage of entries in the open arms of the EPM test. * and # p < 0.05 vs. Veh–Veh and Tumor-Veh, respectively (Fig. A, n = 7–9); * p < 0.05 when compared to Veh–Veh (B, C, n = 6–8). All data are expressed as mean ± S.E.M.
factor: F(1,22) = 0,0006397; p > 0.05]), (Fig. 1B), time spent in the open arms of EPM test [treatment factor: F(1,22) = 1.881; p > 0.05], (Fig. 1C). Moreover, it was not observed statistical difference in the open arm entries [condition factor: F(1,22) = 2.498; p > 0.05; treatment factor: F(1,22) = 0,1045; p > 0.05], (Fig. 1D). It is also important to point out that the findings are not related to a locomotor deficit, since no changes in the number of transitions evaluated in the LDT or in the number of entries into the enclosed arms of the EPM were detected (data not shown). 3.2. Systemic midazolam treatment reduced anxiogenic-like behaviors but failed to attenuate thermal hyperalgesia after inoculation of tumor cells Inoculation of tumor cells caused a significant orofacial heat hyperalgesia on day 6 after the procedure (Fig. 2A, p < 0.05) which was not affected by systemic treatment with midazolam (0.5 mg/kg, Fig. 2A, p > 0.05). In an independent group of animals it was observed an anxiogenic-like behavior in tumor-bearing rats (time in the lit compartment of LDT test [condition factor: F(1,35) = 17.41; p < 0.05], Fig. 2B). Systemic treatment with midazolam was able to induce an anxiolytic-like effect both in the control and tumor-bearing rats assessed 15 min after the treatment [treatment factor: F(1,35) = 9.093, p < 0.05], (Fig. 2B). Despite the statistical analysis do not show effect on the condition factor [F(1,34) = 0,7359; p = 0.397], it was clearly observed an effect on the treatment factor [F(1,34) = 72.83, p < 0.05] and also the interaction between the factors treatment and condition [interaction factor: F(1,34) = 8.976; p < 0.05]. Also, no statistically significant difference was observed on open arm entries [condition factor: F(1,34) = 0,07114; p > 0.05; treatment factor: F(1,34) = 3.382; p > 0.05], (Fig. 1D).
4. Discussion The present data showed that tumor-bearing rats present an anxiogenic-like behavior, in addition to the nociceptive behavior. Local treatment with lidocaine reduced the heat hyperalgesia, without changing the anxiety-like behaviors, while midazolam treatment failed to change the heat hyperalgesia, but induced an anxiolytic-like effect in rats. The facial cancer model used in the present study has been well characterized by our group [19,unpublished observations] and others [20–22]. According to these studies, rats submitted to inoculation of Walker-256B cells into the vibrissa pad display increased spontaneous grooming, thermal and mechanical orofacial hypersensitivity starting as soon as 4 days after cells inoculation and persisting around 10 days. The nociceptive behaviors on this model are not significantly mediated by cancer-induced peripheral inflammation, suggesting the contribution of a neuropathic pain component [21]. Thus, it represents a valuable model in the study of pain mechanisms related to orofacial cancer, as well as in the study of other complications related to this condition. It has been reported that anxiety is a frequent symptom experienced by cancer patients and it is usually under diagnosed and undertreated. Moreover, limited research has been undertaken to understand its mechanism, to establish its appropriate treatment and its relation with pain [23,24]. In the current study we demonstrated that rats with orofacial cancer display nociceptive- and anxiety-like behaviors, which corroborates clinical studies that addressed the major symptoms experienced by cancer pain patients. Additionally, this finding is in agreement with some pre-clinical studies that evaluate nociceptive- and anxietylike behaviors in models of inflammatory and neuropathic pain [18,25–28]. Some of these studies reported that centrally-acting
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Fig. 2. Effect of systemic midazolam treatment in animals submitted to the heat hyperalgesia, elevated plus maze (EPM) and light–dark transition (LDT) tests after tumor cells inoculation. (A) Heat hyperalgesia time course. (B) Percentage of time spent in the lit compartment of the LDT test. (C) Percentage of time spent in the open arms of the EPM test. (D) Percentage of entries in the open arms of the EPM test. * and # p < 0.05 vs. Veh–Veh and Tumor-Veh, respectively, (A, n = 6–10); * p < 0.05 vs. Veh–Veh and # p < 0.05 vs. its respective control group (B, C, n = 8–15). All data are expressed as mean ± S.E.M.
analgesics, such as morphine, tramadol and gabapentin, are able to relieve anxiety-like behaviors in chronic pain models probably via their anti-nociceptive properties [18,29]. Likewise, recent studies have suggested that there are common central sites that play a role in the regulation of pain sensation and anxiety-like behaviors [30,31]. Thus, according to the current literature, anxiolytic and analgesic effects can be simultaneously achieved by targeting specific central sites, such as neurons in the anterior cingulate cortex or in the prelimbic cortex [30,31]. In line with this observation, we demonstrated that peripheral nerve block with lidocaine was able to attenuate heat hyperalgesia in tumor-bearing rats, but did not influence the anxiety-like behaviors. We do not believe that this result is specific to the sensorial modality assessed herein (i.e. heat hyperalgesia), since unpublished observations of our group demonstrated a positive correlation of anxiety-like behaviors and mechanical allodynia in tumor-bearing rats. In addition, we suggest that the lack of effect of lidocaine in anxiety-like behaviors is mainly due to its restriction in the periphery, but it can be also due to the short antihyperalgesic effect promoted by lidocaine. It can be speculated that a longer period of peripheral pain relief would result in attenuation of anxiety-like behaviors. In chronic pain, nociceptors trigger an increased rate of action potentials and subsequently convey pain signals to several brain areas that are thought to be involved in the initiation of pain perception and anxiety [30–32]. Thus, it remains to be investigated if prolonged peripheral pain relief would contribute to diminish the anxiety-related parameters. Several studies have reported that anxiety can affect the perception of pain and enhance pain sensation [33–36]. Benzodiazepines are extensively indicated to treat some types of anxiety disorders, but side effects and dependence limit their long-term use [37,38]. There is no consistent clinical evidence that benzodiazepines can produce analgesic effect, but they are prescribed to chronic pain
patients to relieve anxiety [39,40]. On the other hand, few preclinical studies that have addressed the effect of benzodiazepines on pain models have generated controversial data, which can be related to the dose employed, route and period of administration, as well as pain model [41–43]. According to our data, a single injection of midazolam resulted in a marked anxiolytic-like effect, but failed to modify the heat hyperalgesia in tumor-bearing rats, which is in line with previous observations [44,45]. Indeed, this finding completely corroborates the study by Roeska and colleagues [18], which demonstrated anxiolytic-like, but not anti-hyperalgesic-like, effects of midazolam in rats submitted to chronic constriction injury of the sciatic nerve. One of the main mechanisms related to midazolam-anxiolytic effect is the enhancement of GABAA -mediated inhibition in brain areas such as amygdala, prefrontal cortex, hypothalamus and hippocampus, among others [46–48]. It is interesting to note that alterations in hippocampus neurons have been reported in models of chronic pain, but they have been related to the anxiety-like, but not nociceptive-like, behaviors presented by the animals [26,31]. Thus, it could be suggested that the lack of effect of midazolam on hyperalgesia is related to its action on a brain substrate much more related to anxiety than to pain. In addition, there is evidence that anxiety is a consequence of persistent pain, as morphine and gabapentin reversed anxiety-like behaviors in neuropathic animals without having an effect on anxiety in sham-operated rats [18]. This later study corroborates previous reports showing that pain alleviation reduced the behavioral signs of anxiety [49,50]. In conclusion, our results demonstrated that facial tumorbearing rats display nociceptive and anxiogenic-like behaviors, which are not concomitantly attenuated by brief heat hyperalgesia relief or by decreasing anxiety. However, we can not discard the possibility that these conditions are correlated, as only the
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