Involvement of TRPV1 in Nociceptive Behavior in a Rat Model of Cancer Pain

The Journal of Pain, Vol 9, No 8 (August), 2008: pp 687-699 Available online at www.sciencedirect.com

Involvement of TRPV1 in Nociceptive Behavior in a Rat Model of Cancer Pain Masamichi Shinoda,* Akina Ogino,* Noriyuki Ozaki,* Hiroko Urano,*,‡ Katsunori Hironaka,*,† Masaya Yasui,* and Yasuo Sugiura* *Department of Functional Anatomy and Neuroscience and † Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan; and ‡ Department of Neural Regeneration and Cell Communication, Mie University Graduate School of Medicine, Mie, Japan.

Abstract: To investigate the mechanisms underlying cancer pain, we developed a rat model of cancer pain by inoculating SCC-158 into the rat hind paw, resulting in squamous cell carcinoma, and determined the time course of thermal, mechanical sensitivity, and spontaneous nocifensive behavior in this model. In addition, pharmacological and immunohistochemical studies were performed to examine the role played by transient receptor potential vanilloid (TRPV)1 and TRPV2 expressed in the dorsal root ganglia. Inoculation of SCC-158 induced marked mechanical allodynia, thermal hyperalgesia, and signs of spontaneous nocifensive behavior, which were diminished by systemic morphine administration. Intraplantar administration of the TRPV1 antagonist capsazepine or TRP channels antagonist ruthenium red did not inhibit spontaneous nocifensive behavior at all. However, intraplantar administration of capsazepine or ruthenium red completely inhibited mechanical allodynia and thermal hyperalgesia produced by SCC-158 inoculation. Immunohistochemically, the number of TRPV1-positive, large-sized neurons increased, whereas there was no change in small-sized neurons in the dorsal root ganglia. Our results suggest that TRPV1 play an important role in the mechanical allodynia and thermal hyperalgesia caused by SCC-158 inoculation. Perspective: We describe a cancer pain model that induced marked mechanical allodynia, thermal hyperalgesia, signs of spontaneous nocifensive behavior, and upregulation of TRPV1. Mechanical allodynia and thermal hyperalgesia were inhibited by TRP channel antagonists. The results suggest that TRPV1 plays an important role in the model of cancer pain. © 2008 by the American Pain Society Key words: TRPV1, TRPV2, thermal hyperalgesia, mechanical allodynia, spontaneous nocifensive behavior.

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pproximately 75% to 90% of patients with advanced cancer have chronic pain related to tumor progression.34,39 Basic research on the mechanisms of cancer pain would be enhanced by the development of adequate animal models. Recently, several kinds of cancer pain models have been developed, including

Received February 9, 2007; Revised January 19, 2008; Accepted February 26, 2008. Supported in part by a Grant-in-Aid from the Japan Society for the Promotion of Science. Address reprint requests to Dr. Yasuo Sugiura, Department of Functional Anatomy and Neuroscience, Nagoya University School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya, 466-8550, Japan. E-mail: ysugiura@ med.nagoya-u.ac.jp 1526-5900/$34.00 © 2008 by the American Pain Society doi:10.1016/j.jpain.2008.02.007

pain models for bone cancer,33,53 skin cancer,41 neuropathic cancer,44 and cancer pain associated with squamous cell carcinoma.4,35 These models have demonstrated the distinct pharmacological and neurochemical aspects of cancer pain, suggesting that it is composed of inflammatory, neuropathic, and tumorigenic components.30 The transient receptor potential vanilloid 1 (TRPV1) and its homologue TRPV2, which belong to the TRPV subfamily of the large TRP ion channel super family, are critical contributors to normal and pathological pain.49 TRPV1 and TRPV2 are highly expressed in primary sensory neurons within dorsal root ganglia (DRGs). TRPV1 can be activated by noxious heat (⬎43°C), extracellular acidification, various lipids, and capsaicin and is 687

688 not only observed in small-diameter C-type neurons but also medium-diameter A␦-type neurons associated with the transmission of nociceptive information in DRG.29,50 TRPV1 appears to be upregulated, and its function increases in IB4-positive C-fiber nociceptors under inflammatory conditions.2,5,48 TRPV1 also appears to be upregulated in DRG after nerve injury.12,19,29 Moreover, TRPV1 expression increased in the DRG after cancer infiltration and contributed to thermal hyperalgesia, which is inhibited by TRPV1 antagonist.4 Selective blockade of TRPV1 results in a significant attenuation of nocifensive behaviors in a model of bone cancer pain.14,32 TRPV2 is activated by high temperatures (⬎52°C) and observed in medium- to large-diameter A␦- and A␤-type primary sensory neurons.1,7,24,28 Both TRPV1 and TRPV2 are thought to contribute to nociceptive processing over a range of thermal stimuli because both of these channels are activated at different thermal thresholds and are present and functional in distinct subpopulations of DRG neurons. It is possible that TRPV2, similar to TRPV1, contributes to pathological pain due in particular to cancer infiltration. However, little is known about the involvement of TRPV2 in the cancer pain. In this study, we developed a new model of cancer pain by using a subcutaneous inoculation of squamous cell carcinoma into peripheral tissue, which resulted in spontaneous nocifensive behavior, mechanical allodynia, and thermal hyperalgesia. We determined the time course of pain behavior and performed immunohistochemical and pharmacological studies to examine the role played by TRP channels expressed in the DRG. The altered expression of TRP channels may be involved in the nociceptive behavior observed in this model.

Materials and Methods Experimental Animals Forty-seven male Fisher rats (Nihon SLC, Hamamatsu, Japan) weighing 200 to 300 g were used in this study. They were exposed to a light-dark cycle (12:12-hour) and kept in a temperature-controlled room (23°C). This study was conducted under the auspices of the local animal ethics committee in accordance with the Guidelines for Animal Experiments of the Nagoya University Graduate School of Medicine, the Animal Protection and Management Law of the Japanese Government (No. 105), and the guidelines of the International Association for the Study of Pain.56

Inoculation of Tumor Cells and Measurement of Tumor Growth The SCC-158 cells (squamous cell carcinoma derived from the external acoustic meatus of the Fisher rat) were provided by the Japanese Cancer Research Resources Bank (JCRB, Tokyo, Japan). The tumor cells were prepared for the inoculation as previously described.35 After the induction of general anesthesia with sodium pentobarbital (Nembutal; Abbot Laboratories, Chicago, IL) (50 mg/kg i.p.), 35 rats were subcutaneously injected

TRPV1 and TRPV2 Involvement in Cancer Pain with a 50-␮L solution of the SCC-158 cells (2 ⫻ 107 cells) in the plantar region of the hind paw and 50 ␮L of 0.1 M phosphate-buffered saline (PBS) in the contralateral hind paw. In 12 rats, PBS was subcutaneously injected into the bilateral hind paw. After the injection, the rats were given laboratory chow and tap water ad libitum under conventional laboratory conditions. To assess tumor growth, the thickness of the paw (dorsal to plantar thickness) was measured before tumor inoculation to assess basal values. The same procedure was repeated on days 4, 8, 12, 17, 20, and 24 after tumor inoculation.

Behavioral Assay The analyses of the behavioral experiments were made by the experimenters, who were blinded to the experimental conditions. Measurements of spontaneous nocifensive behavior, mechanical allodynia, and thermal hyperalgesia were performed for 24 days after tumor inoculation during the experimental period. Effects of morphine, capsazepine, and ruthenium red were measured on days 20 through 24 after tumor inoculation. Animals were placed in a plastic cage (17 ⫻ 22 ⫻ 14 cm) where, after a 10-minute adaptation interval, their spontaneous nocifensive behaviors were measured over the following 10-minute period. A nociceptive score was assigned by adapting the classic method of measuring nociceptive responses to the injection of formalin.33 Thus, a score of 0 was assigned when the paw was in normal contact with the floor; 0.5 when the paw was flinched; 1 when the paw was lifted; and 2 when the paw was licked. The numbers of each behavior during the observation time (600 seconds) were counted and were multiplied by their respective scores. The final score was determined by the summation of the multiplied respective scores. Animals were tested before tumor inoculation to assess basal values. The same procedure was performed on days 3, 6, 9, 14, 15, 18, 21, and 24 after tumor inoculation. The mechanical sensitivity of the plantar surface of the paw was assessed by using the Dynamic Plantar Aesthesiometer (DPA; Ugo Basile, Comerio, Italy) Animals were placed individually in a small enclosed testing arena (20 ⫻ 18 ⫻ 14 cm) with a wire mesh floor and allowed to acclimate to their surroundings for a minimum of 20 minutes before testing. The filament of the DPA device was placed under the paw, where it was raised and the force progressively increased until the animal withdrew the paw or until the force reached a maximum of 50 g. The DPA automatically recorded the force at which the foot was withdrawn and the withdrawal latency. A 5-minute interval was used between consecutive stimulations of the same paw. Each paw was tested 5 times, and the withdrawal thresholds for each paw were averaged. The same procedure was performed on days 3, 6, 9, 14, 15, 18, 21, and 24 after tumor inoculation. Responses to noxious radiant heat were determined with the method of Hargreaves, using the Plantar test (Ugo Basile).15 Rats were allowed to acclimate for 30 minutes in a transparent plastic chamber. Withdrawal

Shinoda et al latencies were defined as the elapsed time between activation of the heat source and the paw withdrawal. A cutoff of 15 seconds was established to avoid tissue damage. A 5-minute interval was used between consecutive stimulations of the same paw. Each paw was tested 5 times, and the latencies for each paw were averaged. Animals were tested before tumor inoculation to assess basal values. The same procedure was performed on days 3, 6, 9, 14, 15, 18, 21, and 24 after tumor inoculation.

Intraperitoneal Administration of Morphine Animals in the tumor inoculation group were tested with morphine to see whether alteration in the nociceptive scores, paw withdrawal thresholds, or paw withdrawal latencies reflected spontaneous pain behavior, mechanical allodynia, or thermal hyperalgesia, respectively. Morphine was dissolved in physiological saline. On days 20 through 24 after the inoculation of tumor cells, morphine (7.5, 10, and 15 mg/kg for testing nociceptive scores and paw withdrawal thresholds; 7.5, 10 mg/kg for testing alteration of paw withdrawal latencies) was injected intraperitoneally in a final volume of 0.5 mL. Control animals received an equivalent volume of vehicle. Nociceptive scores, paw withdrawal thresholds, and paw withdrawal latencies were determined both before and 30 minutes after morphine treatment in the tumor-inoculated paw.

Peripheral Administration of TRPV1 and TRP Channel Antagonists In the tumor-inoculated paw, to assess the involvement of TRPV1 and other TRP channels in the spontaneous nocifensive behavior, mechanical allodynia, and thermal hyperalgesia induced by tumor cell inoculation, animals were treated with the TRPV1 antagonist capsazepine (Biomol International, Plymouth Meeting, PA) and the TRP channels antagonist ruthenium red (Sigma, St. Louis, MO). Capsazepine was dissolved in DMSO (10% in saline), and ruthenium red was dissolved in physiological saline. On days 20 through 24 after the inoculation of tumor cells, capsazepine (50, 100 pmol/paw) or ruthenium red (1.2, 120 nmol/paw) was subcutaneously injected into the tumor-inoculated paw in a final volume of 50 ␮L. Injections were made with a 30-gauge needle, using a 50-␮L Hamilton syringe. Doses of antagonists were determined from previous reports.40,54 Nociceptive scores, paw withdrawal thresholds, and paw withdrawal latencies were determined both before and 30 minutes after treatment with antagonists. Control animals received an equivalent volume of vehicle.

Tissue Preparation On days 14 and 24 after tumor cell inoculation, rats were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg; Nembutal; Abbot Laboratories) and transcardially perfused with heparinized saline followed by a cold fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).

689 Bilateral L5 DRGs and the tumor-inoculated paw were immediately dissected out after perfusion and immersed in the same fixative for 4 hours at 4°C. Postfixed DRGs and the tumor-inoculated paw were kept in 0.1 M PBS containing 20% sucrose for cryoprotection. The specimens were then embedded in Tissue Tek (Sakura Finetechnical, Tokyo, Japan) and stored until cryosection at ⫺20°C. The DRGs were cut on the horizontal plane along the long axis of the ganglia on a cryostat at a thickness of 10 ␮m. Every 20th section was chosen, yielding approximately 5 sections per DRG from each animal. The tumorinoculated paw was cut on the vertical plane along its long axis on a cryostat at a thickness of 10 ␮m or 30 ␮m. After rinsing with 0.1 M PBS, 10-␮m sections of the tumor-inoculated paw were stained with hematoxylin and eosin. Sections were mounted on MAS-coated glass slides (Matsunami, Tokyo, Japan) and dried at room temperature overnight.

Immunohistochemistry For the immunohistochemistry of DRGs, rabbit polyclonal antisera against synthetic mice TRPV1 (Transgenic Inc., Kumamoto, Japan) and TRPV2 (Sigma) were used in this study. Each antibody was diluted at a concentration of 1:1000 in 0.1 M PBS containing 4% normal goat serum and 0.3% Triton-X 100 (Sigma). Sections of the DRGs were incubated in a solution of either TRPV1 or TRPV2 antiserum for 3 days at 4°C. For the immunohistochemistry of the tumor-inoculated paw, anti-PGP-9.5 rabbit IgG against synthetic rat PGP-9.5 (Ultra Clone, Isle of Wight, UK) was used as primary antibody. The antibody was diluted at a concentration of 1:1000 in 0.1 mol/L PBS containing 4% normal goat serum and 0.3% Triton-X 100 (Sigma). After rinsing with 0.1 M PBS, sections were reacted with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at a dilution of 1:200 in 0.1 M PBS for 2 hours at room temperature. After rinsing with 0.1 M PBS, they were immersed in a solution of avidin and biotin-peroxidase complex (Vector Laboratories) at a dilution of 1:100 in 0.1 M PBS for 90 minutes at room temperature. The sections were then immersed in PBS containing 0.1% 3, 3=-diaminobenzidine dihydrochloride (Sigma). Antibody-binding sites were visualized by adding 0.004% hydrogen peroxide. Sections were examined and photographed with a light microscope equipped with digital camera. Immunoreactive and negative cells in the DRGs were manually traced with a light microscope– equipped, computer-aided image analyzing system (NeuroLucida; MicroBrightfield, Colchester, VT) that automatically calculated the total number and diameters of DRG cells. Immunoreactive cells were classified by cell diameter at 5-␮m intervals. The ratio of immunoreactive cells in each animal was calculated by the formula: (100 ⫻ total number of immunoreactive cells in 5 sections of DRG in each classified group/total number of cells in 5 sections of DRG). The calculation was performed on each group of cells classified by cell diameter.

690 No specific labeling was observed in the absence of primary antibody.

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The data are expressed as mean ⫾ SEM. Results were analyzed by using 1-way analysis of variance (ANOVA) followed by Holm-Sidak multiple comparison test or a 2-way repeated-measures ANOVA, followed by Bonferroni multiple comparison tests where appropriate. A difference was considered statistically significant at P ⬍ .05.

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General Observation and Tumor Growth General features of the tumor cell–inoculated rats were well maintained for 28 days. Signs of tumor growth, observed as a swelling around the inoculated site, became visible in most animals by day 7. Macroscopic examinations revealed apparent metastasis to the lung but not to the other organs on day 24. The thickness of the tumor-inoculated paws significantly increased on day 4 after inoculation compared with PBS-injected paws without tumor inoculation, reaching mean heights of 5.5 ⫾ 0.2 mm compared with the PBS means of 4.9 ⫾ 0.0 mm. The mean thickness of the tumor-inoculated paws was significantly greater than those of the PBS-injected paws on day 4 and throughout the rest of the experimental period (Fig 1).

Microscopic Observation of Peripheral Structure in the Cancer Region On day 14 in the tumor-inoculated paw, a tumoral mass was present at the site of inoculation and had displaced nearby tissues. The tumor was surrounded by infiltrated inflammatory and immune cells and encapsulated by a thin connective tissue sheet so that the

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Figure 2. Microscopic observation of the tumor-inoculated paw on day 14. (A) Photomicrograph of the tumor and its surrounding tissue. T indicates tumor; I, infiltration of inflammatory cells; C, connective tissue capsule; SC, subcutaneous tissue; D, dermis; E, epidermis. Size scale bar, 500 ␮m. (B) Photomicrograph of the nerve surrounded by the tumor cells. N indicates nerve. Size scale bar, 100 ␮m. (C) PGP-9.5-immunoreactive fibers (arrows) in the tumor-inoculated paw. Size scale bar, 100 ␮m.

Figure 1. Changes in thickness of the paw followed by inoculation with SCC-158. Rats were given a subcutaneous injection of SCC-158 cells or phosphate-buffered saline (PBS) into the hind paw. ● indicates ipsilateral paw of tumor-inoculated animals; Œ, contralateral paw of tumor-inoculated animals with PBS injection; ‘, paw of PBS-injected animals without tumor inoculation. Each point indicates mean ⫾ SEM. *P ⬍ .05 compared with PBS-injected paw on the same day (2-way ANOVA with repeated measures followed by Bonferroni multiple comparison tests).

surrounding tissues such as bones, tendons, and muscles were not destroyed (Fig 2A). Although tumor cells did not infiltrate, they surrounded and made direct contact with peripheral nerves (Fig 2B). PGP-9.5-immunoreactive (ir) fibers were found in the epidermis, dermis, subcuta-

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neous tissues, and occasionally in the connective tissue capsule around the tumor, though there are no nerve fibers in the tumoral mass and capsule (Fig 2C).

Spontaneous Nocifensive Behavior There were significant differences in the incidence of flinching, licking, or lifting between the tumor-inoculated and PBS-injected paws without tumor inoculation on days 3, 18, 21, and 24 after inoculation (Fig 3A). The mean nociceptive score of the contralateral paw of tumor-inoculated animals with PBS injection tended to be higher than that of PBS injected paw without tumor inoculation but did not reach significance. Effects of morphine were tested on day 23 and 24 after tumor inoculation. Intraperitoneal morphine (7.5 mg/kg) significantly reduced the nociceptive score of tumor-in-

Figure 4. Effects of antagonists on spontaneous nocifensive behavior during perambulation of animals inoculated with SCC158 cells. Values of histograms are represented as mean ⫾ SEM. (A) Effects of intraplantar capsazepine (CPZ; 50 and 100 pmol per paw) injections on spontaneous nocifensive behavior days 21 to 24 after SCC-158 inoculation. (B) Effects of intraplantar ruthenium red (RR; 1.2 and 120 nmol per paw) injections on spontaneous nocifensive behavior days 21 to 24 after SCC-158 inoculation. NAT indicates no antagonist treatment. Data were analyzed by using a 1-way ANOVA followed by Holm-Sidak multiple comparison test. *P ⬍ .05.

Figure 3. Changes in spontaneous nocifensive behavior of animals inoculated with SCC-158 cells. (A) Time course of spontaneous nocifensive behavior during perambulation of animals subcutaneously injected into the hind paw with either SCC-158 cells or phosphate-buffered saline (PBS). ● indicates ipsilateral paw of tumor-inoculated animals; Œ, contralateral paw of tumor-inoculated animals with PBS injection; ‘, paw of PBS-injected animals without tumor inoculation. Each point indicates mean ⫾ SEM. *P ⬍ .05 compared with PBS-injected paw without tumor inoculation on the same day (2-way ANOVA with repeated measures followed by Bonferroni multiple comparison tests). (B) Effect of intraperitoneal morphine (7.5, 10, and 15 mg/kg) on spontaneous nocifensive behavior of tumor-inoculated animals days 21 to 24 after inoculation. NMT indicates no morphine treatment. Values of histograms are represented as mean ⫾ SEM. Data were analyzed by using 1-way ANOVA followed by Holm-Sidak multiple comparison test. *P ⬍ .05.

oculated paws (0.6 ⫾ 0.6) compared with vehicle treatment (4.9 ⫾ 1.6), administration of which was not effective (Fig 3B). The effects of antagonists were tested on days 21 through 24 after tumor inoculation. In the tumor-inoculated paw, intraplantar administration of the TRPV1 antagonist capsazepine (50 and 100 pmol per paw) had no effect on nociceptive scores compared with the vehicle (Fig 4A). Intraplantar administration of TRP channel antagonist ruthenium red (1.2 and 120 nmol per paw) was also ineffective compared with the vehicle (Fig 4B), nor did capsazepine or ruthenium red affect the nociceptive scores of PBS-injected paws. In addition, no other overt behavioral effects such as motor defects or sedation were observed (data not shown).

Mechanical Allodynia The withdrawal thresholds of tumor-inoculated paws showed a significant decrease on days 3, 6, 14, 18, 21, and 24 after inoculation compared with PBS-injected paws

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without tumor inoculation, indicating mechanical allodynia (Fig 5A). Effects of morphine were tested on days 23 and 24 after tumor inoculation. Intraperitoneal morphine (7.5 mg/kg) increased withdrawal thresholds of tumor-inoculated paws (40.1 ⫾ 1.3 g), whereas vehicle treatment was not effective (Fig 5B). Effects of antagonists were tested on days 21 through 24 after tumor inoculation. Capsazepine (50 and 100 pmol per paw) produced a marked reversal of mechanical allodynia on tumor-inoculated paws (Fig 6A). Ruthe-

Figure 6. Effects of antagonists on mechanical sensitivity of animals inoculated with SCC-158 cells. Values of histograms are represented as mean ⫾ SEM. (A) Effect of intraplantar capsazepine (CPZ; 50 and 100 pmol per paw) injections on mechanical sensitivities of days 21 to 24 after SCC-158 inoculation. (B) Effects of intraplantar ruthenium red (RR; 1.2 and 120 nmol per paw) injections on mechanical sensitivities of days 21 to 24 after SCC-158 inoculation. Black indicates tumor-inoculated paw; gray, PBS-injected paw; NAT, no antagonist treatment. Data were analyzed by using a 1-way ANOVA followed by HolmSidak multiple comparison test. *P ⬍ .05.

Figure 5. Changes in mechanical sensitivity measured in the paw. (A) Time course of withdrawal thresholds of animals injected subcutaneously with SCC-158 cells and phosphate-buffered saline (PBS) into the hind paw. ● indicates ipsilateral paw of tumor-inoculated animals; Œ, contralateral paw of tumorinoculated animals with PBS injection; ‘, paw of PBS-injected animals without tumor inoculation. Each point indicates mean ⫾ SEM. *P ⬍ .05 compared with PBS-injected paw without tumor inoculation on the same day (2-way ANOVA with repeated measures followed by Bonferroni multiple comparison tests). (B) Effects of intraperitoneal morphine (7.5, 10, and 15 mg/kg) injections on withdrawal thresholds of tumor-inoculated paw of days 21 to 24 after inoculation. Black indicates tumor-inoculated paw; gray, PBS-injected paw; NMT, no morphine treatment. Values of histograms are represented as mean ⫾ SEM. Data were analyzed by using a 1-way ANOVA followed by HolmSidak multiple comparison test. *P ⬍ .05.

nium red (1.2 and 120 nmol per paw) also significantly inhibited mechanical allodynia induced by tumor cell inoculation (Fig 6B). Therefore, capsazepine and ruthenium red produced a complete inhibition of mechanical allodynia induced by tumor cell inoculation while exerting no effects on paw withdrawal thresholds of PBS-injected paws. No other overt behavioral effects such as motor defects or sedation were observed (data not shown).

Thermal Hyperalgesia The paw withdrawal latencies of tumor-inoculated paws assessed by the plantar test showed significant decreases on days 6, 18, 21, and 24 after inoculation compared with the PBS-injected paw. The decreases of the paw withdrawal latencies indicate the induction of thermal hyperalgesia. No significant changes were observed

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in the withdrawal latencies of the PBS-injected paws of animals with or without tumor inoculation throughout the experimental period (Fig 7A). Effects of morphine were tested on days 23 and 24 after tumor inoculation. Intraperitoneal morphine (10 mg/kg) increased withdrawal latencies on tumor-inoculated paws (14.5 ⫾ 0.3 seconds). Vehicle treatment proved to be ineffective (Fig 7B). Effects of antagonists were tested on days 21 through 24 after tumor inoculation. Capsazepine (100 pmol per paw) and ruthenium red (1.2 and 120 nmol per paw) produced a marked reversal of thermal hyperalgesia on tumor-inoculated paws (Fig 8A and B). Therefore, cap-

Figure 8. Effects of antagonists on heat sensitivity of animals inoculated with SCC-158 cells. Values of histograms are represented as mean ⫾ SEM. (A) Effects of intraplantar capsazepine (CPZ; 50 and 100 pmol per paw) injection on heat sensitivity of days 20 to 24 after SCC-158 inoculation. (B) Effects of intraplantar ruthenium red (RR; 1.2 and 120 nmol per paw) injections on heat sensitivity of days 21 to 24 after SCC-158 inoculation. Black indicates tumor-inoculated paw; gray, phosphate-buffered saline (PBS)-injected paw; NAT, no antagonist treatment. Data were analyzed by using a 1-way ANOVA followed by Holm-Sidak multiple comparison test. * P ⬍ .05.

Figure 7. Changes in heat sensitivities measured in the paw. (A) Time course of withdrawal latencies of animals inoculated with SCC-158 cells. ● indicates ipsilateral paw of tumor-inoculated animals; Œ, contralateral paw of tumor-inoculated animals with phosphate-buffered saline (PBS) injection; ‘, paw of PBSinjected animals without tumor inoculation. Each point indicates mean ⫾ SEM. *P ⬍ .05, compared with PBS-injected paw without tumor inoculation on the same day (2-way ANOVA with repeated measures followed by Bonferroni multiple comparison tests). (B) Effects of intraperitoneal morphine (7.5 and 10 mg/kg) injections on withdrawal latencies of tumor-inoculated paw of days 21 to 24 after inoculation. Black indicates tumorinoculated paw; gray, PBS-injected paw; NMT, no morphine treatment. Values of histograms are represented as mean ⫾ SEM. Data were analyzed by using a 1-way ANOVA followed by Holm-Sidak multiple comparison test. *P ⬍ .05.

sazepine and ruthenium red produced complete inhibitions of thermal hyperalgesia induced by tumor cell inoculation. Neither capsazepine nor ruthenium red had any effect on paw withdrawal latencies of PBS-injected paws. Motor defects or sedation were not observed in the experimental period (data not shown).

Alterations of TRPV1 and TRPV2 Expressions in Dorsal Root Ganglion After determining the immunohistochemistry for TRPV1 and TRPV2, immunoreactive cells were stained dark or light brown so as to be readily distinguished from the nonpositive cells (arrows in Fig 9A–F). Twenty-four days after tumor cell inoculation into the paw, 34.8% ⫾ 5.4% of neurons were TRPV1-positive in the DRG ipsilateral to the tumorinoculated paw, whereas 38.2% ⫾ 3.6% of neurons were TRPV1-positive in DRGs contralateral to the tumor-inocu-

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Figure 9. Photomicrographs of TRPV1 (A, C, E) and TRPV2 (B, D, F) immunoreactive cells (arrows) in the dorsal root ganglia (DRG) (L5) 24 days after tumor inoculation. (A, B), DRG in animals injected with phosphate-buffered saline (PBS) bilaterally without tumor. (C, D), DRG ipsilateral to tumor inoculation. (E, F), DRG contralateral to tumor inoculation. Size scale bar, 100 ␮m.

lated paw. In animals injected with PBS bilaterally without tumor cell inoculation, 30.8% ⫾ 4.2% of neurons were TRPV1-positive. There was no significant difference in the ratio of TRPV1-positive cells among DRGs ipsilateral to the tumor-inoculated paw, contralateral to the tumor-inoculated paw, and in animals treated with PBS alone. TRPV1-positive cells were distributed in various sizes (ipsilateral to tumor inoculation: Range ⫽ 10.8 – 87.7 ␮m, mean ⫾ SEM ⫽ 37.8 ⫾ 0.7 ␮m; contralateral to tumor injection: Range ⫽ 13.6 –81.4 ␮m, mean ⫾ SEM ⫽ 35.3 ⫾ 1.3 ␮m; DRG in animals treated with PBS alone: Range ⫽ 12.3– 66.2 ␮m, mean ⫾ SEM ⫽ 32.3 ⫾ 1.0 ␮m). The cell diameter analysis revealed that the TRPV1positive cells in DRGs ipsilateral to the tumor-inoculated paw increased strikingly in cell groups above 40 ␮m in diameter compared with DRGs in animals treated with PBS alone. Interestingly, the TRPV1-positive cells in DRGs contralateral to the tumor-inoculated paw, which were ipsilateral to PBS-injected paws also increased in cell groups 40 to 50 ␮m in diameter, compared with animals treated with PBS alone (Fig 10A). Twenty-four days after tumor cell inoculation into the paw, 17.8% ⫾ 1.5% of neurons were TRPV2-positive in DRGs ipsilateral to the tumor-inoculated paw. On the other hand, 8.7% ⫾ 3.4% of neurons were TRPV2-positive in DRGs contralateral to the tumor-inoculated paw. In

animals injected with PBS bilaterally without tumor cells, 11.3% ⫾ 1.5% of neurons were TRPV2-positive. There was no significant difference in the ratio of TRPV2-positive cells among DRGs ipsilateral to the tumor-inoculated paw, contralateral to the tumor-inoculated paw and in animals treated with PBS alone. TRPV2-positive cells were distributed in various sizes (ipsilateral to tumor inoculation: Range ⫽ 15.1–78.8 ␮m, mean ⫾ SEM ⫽ 37.4 ⫾ 2.0 ␮m; contralateral to tumor inoculation: Range ⫽ 12.7–75.6 ␮m, mean ⫾ SEM ⫽ 36.5 ⫾ 3.0 ␮m; DRG in animals treated with PBS alone: Range ⫽ 14.6 – 63.5 ␮m, mean ⫾ SEM ⫽ 35.1 ⫾ 1.3 ␮m). In contrast to TRPV1, the TRPV2-positive cells in DRGs ipsilateral to the tumor-inoculated paw increased only in the group 55 to 60 ␮m in cell diameter compared with DRGs in animals treated with PBS alone (Fig 10B).

Discussion SCC-158-Induced Spontaneous Nocifensive Behavior, Mechanical Allodynia, and Thermal Hyperalgesia In this study, tumor growth after the inoculation of SCC-158 cells into the paws caused changes in the me-

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Figure 10. Size-frequency histogram illustrating distribution of the TRPV1- (A) and TRPV2-immunoreactive cells (B) in the dorsal root ganglia (DRGs) (L5) 24 days after subcutaneous inoculation of tumor cells into the paw. Black indicates DRGs ipsilateral to tumor inoculation; white, DRGs contralateral to tumor injection; gray, DRGs in animals injected with phosphate-buffered saline (PBS) bilaterally without tumor cell inoculation. Values of histograms are represented as mean ⫾ SEM. Data were analyzed by using a 1-way ANOVA followed by Holm-Sidak multiple comparison test. *P ⬍ .05.

chanical and heat sensitivities of paws as well as signs of spontaneous pain. In the tumor-inoculated paw, a tumoral mass was surrounded by infiltrated inflammatory cells. The tumor cells made direct contact with peripheral nerves. PGP9.5-ir fibers were found in the epidermis, dermis, and subcutaneous tissues, though there are no fibers in the tumoral mass. These observations support the concept that cancer pain has inflammatory, neuropathic, and tumorgenic components. The nociceptive scores of tumor-inoculated paws showed significant increases on day 18 and beyond compared with PBS-injected paws. Generally, the nociceptive score that quantifies spontaneous nocifensive behaviors in the tumor-inoculated paws showed a time-dependent increase. These data were basically similar to those in our previous study of a mouse model of cancer pain.4,35 These data were also similar to the previous studies of bone cancer pain, which measured other parameters of spontaneous pain behavior, such as spontaneous flinching and guarding.16,17,26 We showed that increased nociceptive scores on tumor-inoculated paws were reversed to the naive value by intraperitoneal morphine. The fact reinforces the suggestion that the spontaneous behaviors we measured are indeed pain related. Similarly, in

humans, morphine reduced severe cancer-induced pain.21,27,38 These findings suggest that the increased nociceptive scores reflect spontaneous pain symptoms induced by the inoculation of squamous cell carcinoma cells (SCC-158) into the paw. In tumor-inoculated paws, mechanical allodynia and thermal hyperalgesia became significant after the inoculation of tumor cells and were maintained throughout the experimental period. Mechanical allodynia and thermal hyperalgesia were inhibited by intraperitoneal morphine. These findings suggested that an infiltration of cancer by an inoculation of squamous cell carcinoma cells (SCC-158) into the paw induced mechanical allodynia and thermal hyperalgesia. Previous studies have proven the usefulness of inoculating tumor cells to characterize the properties of the nociceptive reactions induced by malignant tumors.4,33,35,41,42,44,53 The results of these studies have revealed that different cancer models might have different underlying pain mechanisms based on animal species, tumor types, and locations. The present study demonstrates that a squamous cell carcinoma cell (SCC-158) inoculation into the paws of male Fisher rats produces progressive thermal hyperalgesia, mechanical allodynia, and spontaneous pain behavior. Successful establishment of

696 a new animal model of cancer pain featuring cancer pain in humans is very important to better understand the mechanisms of cancer pain.

Involvement of TRPV Channels in Cancer Pain In this study, intraplantar administration of either the TRPV1 antagonist capsazepine or the TRP channels antagonist ruthenium red resulted in insensitivity to spontaneous pain observed after tumor inoculation. Our results indicate that TRP channels on the periphery do not play an important role in the spontaneous pain behavior observed. Given the inhibitory effect of TRP channel antagonists on both mechanical allodynia and thermal hyperalgesia, the spontaneous pain behavior observed after tumor inoculation is not the result of allodynia or hyperalgesia but rather the continuous activation of nociceptive systems by the tumor. Administration of A-425619, a selective TRPV1 antagonist that does not readily enter the central nervous system (CNS), did not affect either heightened levels of spontaneous wide dynamic range firing in complete Freund’s adjuvantinduced inflammation31 or nocifensive behaviors during the persistent phase of the formalin assay.18 These outcomes are consistent with our data suggesting an absence of peripheral TRPV1’s role in spontaneous pain. In contrast to our results, reduced ongoing pain-related behavior has been observed after TRPV1 antagonist treatment in a mouse model of bone cancer pain.14 Such a result reinforces the idea that the different cancer models might have different underlying mechanisms. Spontaneous pain behavior induced by cancer cell inoculation in our model may be caused by other factors such as ATP and its receptor P2X3. ATP released from tumor cells has been previously reported.6 Increased expression of P2X3 in sensory neurons has been observed in animal models of cancer pain.14 In addition to the periphery, central mechanisms may also be involved in the spontaneous pain behavior observed in this model. In the present study, capsazepine produced a complete reversal of mechanical allodynia on tumor-inoculated paws. In the periphery, TRPV1 is expressed in sensory neurons sensitive to noxious heat (⬎43°C), low pH, and capsaicin.49 No direct link has previously been established between mechanical transduction and TRPV1 activation in vitro. It is known that activation of TRPV1 by capsaicin causes both thermal and mechanical hyperalgesia in rats, primates, and humans.22,45,51 It has been shown that an intradermal capsaicin injection facilitates the responses of dorsal horn neurons due to the input of low-threshold mechanoreceptors and nociceptors.22,46 Thus, in tumor-inoculated paws, a local pH decrease or any other stimuli may activate TRPV1, sensitizing dorsal horn neurons and decreasing the threshold at which mechanical stimuli detect noxious stimuli. TRPV1 receptors have been found in both the peripheral nervous system and the CNS.52 The role of TRPV1 receptors in the CNS in mechanical allodynia has been suggested.11 Although we can not completely exclude the possible central ef-

TRPV1 and TRPV2 Involvement in Cancer Pain fects of capsazepine and ruthenium red, a small amount of these peripherally administered antagonists (pmol per paw) sufficiently reduced mechanical allodynia, suggesting that the effects produced by TRPV1 antagonists are peripherally TRPV1-mediated. The ability of TRP channel antagonists to reduce mechanical hyperalgesia after tumor inoculation may be attributed in part to an altered expression of TRPV1. Tumor inoculation results in increased TRPV1 immunoreactivity in large DRG neurons (above 40 ␮m in cell diameter), which provides evidence for the involvement of TRPV1 in mechanical sensitivity after tumor inoculation and supplies a further rationale for why TRP channel antagonists are effective in reversing mechanical hyperalgesia. TRPV1 antagonists (eg, capsazepine, BCTC, AMG 9810, A-425619, A-784168, and A-795614) have been reported to reduce mechanical allodynia associated with both chronic inflammation and neuropathy in rats11,13,18,37,54 in which an upregulation and/or sensitization of TRPV1 was reported.19,36 A recent report demonstrated that the TRPV1 antagonist JNJ-17203212 inhibited the mechanical hyperalgesia elicited by the development of a femoral mice osteosarcoma.14 The ability of capsazepine to reduce mechanical hyperalgesia after tumor inoculation may be attributed to the tumor-related mechanisms of TRPV1 upregulation or sensitization. Our overall results suggest that the upregulation of TRPV1 in large DRG neurons may play an important role in the mechanical allodynia observed in our rat model of cancer pain. The paw withdrawal latencies under radiant heat showed significant decreases after the inoculation of tumor cells, indicating the induction of thermal hyperalgesia. The TRPV1 antagonist capsazepine produced a complete inhibition of thermal hyperalgesia induced by tumor cell inoculation. TRPV1-positive large DRG neurons increased strikingly after tumor inoculation. These findings suggest that the upregulation of TRPV1 in large DRG neurons plays an important role in the thermal hyperalgesia in this model. The large DRG neurons that innervate A␤-afferents are not normally associated with nociception.23 In pathological conditions such as tumor infiltration, A␤-afferents may come to be activated by noxious heat stimuli through increased TRPV1. Mechanical allodynia and thermal hyperalgesia induced by tumor cell inoculation were also inhibited by TRP channel antagonist ruthenium red. The results presume that the upregulation and/or sensitization of TRP channel receptors other than TRPV1 may also play an important role in the mechanical allodynia and thermal hyperalgesia. Our current data show that TRPV2 increased only in 55- to 60-␮m-sized DRG neurons. Although further rigorous experiments were needed, TRPV2 may be less involved in cancer pain than TRPV1. Recent studies have suggested that TRPV3 and/or TRPV4 in keratinocytes might participate in temperature sensation.8-10 Moreover, mechanosensation is defective in mice lacking TRPV4.25,47 In addition to TRPV1, TRPV2, TRPV3, and TRPV4 may also be involved in nociceptive behavior in our model.

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In DRGs contralateral to the tumor inoculation, TRPV1 markedly increased in the large neurons. The mechanisms by which tumor development induces an increase of TRPV1 in contralateral DRG neurons remain unclear. Several investigators have identified close relationships between nerve growth factor (NGF) and expression of TRPV1.3,20,55 NGF is overexpressed in several types of cancer, including squamous cell carcinoma. The number of TRPV1-expressing neurons in DRGs was increased significantly after intraplantar injection of NGF.43 One possibility is that NGF and/or other substances released from cancer cells (or other cells) may induce phenotypic changes in nerve fibers, thus systemically altering their response characteristics. In conclusion, we have developed a new rat model of

cancer pain by a subcutaneous inoculation of squamous cell carcinoma into the paws that resulted in spontaneous pain behavior, thermal hyperalgesia, and mechanical allodynia. We have shown that TRPV1-expressed neurons increased in the DRG. In addition, we demonstrated the functional significance of TRP channels including TRPV1 in the mechanical allodynia and thermal hyperalgesia induced by tumor cell inoculation. Upregulation and/or sensitization of TRPV in the sensory neurons after tumor cell inoculation may facilitate the transmission of nociceptive information, thus contributing to the resultant pain response. TRP channels including TRPV1 may be a therapeutic target for treating the pain associated with cancer infiltration.

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