Scholarly article on topic 'Vincristine-induced peripheral neuropathic pain and expression of transient receptor potential vanilloid 1 in rat'

Vincristine-induced peripheral neuropathic pain and expression of transient receptor potential vanilloid 1 in rat Academic research paper on "Biological sciences"

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{Vincristine / TRPV1 / "TRPV1 antagonist" / "Dorsal root ganglion" / "Peripheral neuropathic pain"}

Abstract of research paper on Biological sciences, author of scientific article — Terumasa Chiba, Yusuke Oka, Hiroya Sashida, Toshie Kanbe, Kenji Abe, et al.

Abstract The clinical anti-cancer efficacy of vincristine is limited by the development of dose-dependent peripheral neuropathy. Up-regulation of transient receptor potential vanilloid 1 (TRPV1) is correlated with peripheral neuropathy following anti-cancer drug treatment. To analyze the contribution of TRPV1 to the development of vincristine-induced mechanical allodynia/hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after vincristine treatment. Mechanical allodynia/hyperalgesia was tested with von Frey filaments 14 days after intraperitoneal administration of 0.1 mg/kg vincristine in rats. TRPV1 expression in DRGs following vincristine treatment was assessed with western blot analysis and in situ hybridization histochemistry. Vincristine-induced mechanical allodynia/hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3 mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30 mg/kg, s.c.). Vincristine treatment increased the expression of TRPV1 protein in DRG neurons. In situ hybridization histochemistry revealed that most of the TRPV1 mRNA-labeled neurons in the DRG were small in size. Immunohistochemistry showed that isolectin B4-positive small DRG neurons co-expressed TRPV1 protein 14 days after treatment. These results suggest that vincristine treatment increases TRPV1 expression in small DRG neurons. TRPV1 expression may contribute to the development of vincristine-induced painful peripheral neuropathy.

Academic research paper on topic "Vincristine-induced peripheral neuropathic pain and expression of transient receptor potential vanilloid 1 in rat"

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Vincristine-induced peripheral neuropathic pain and expression of transient receptor potential vanilloid 1 in rat

Terumasa Chiba, Yusuke Oka, Hiroya Sashida, Toshie Kanbe, Kenji Abe, Iku Utsunomiya, Kyoji Taguchi

PII: S1347-8613(17)30044-0

DOI: 10.1016/j.jphs.2017.03.004

Reference: JPHS 338

To appear in: Journal of Pharmacological Science

Received Date: 31 October 2016 Revised Date: 17 February 2017 Accepted Date: 15 March 2017

Please cite this article as: Chiba T, Oka Y, Sashida H, Kanbe T, Abe K, Utsunomiya I, Taguchi K, Vincristine-induced peripheral neuropathic pain and expression of transient receptor potential vanilloid 1 in rat, Journal of Pharmacological Science (2017), doi: 10.1016/j.jphs.2017.03.004.

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Vincristine-induced peripheral neuropathic pain and expression of transient receptor potential vanilloid 1 in rat

Terumasa Chiba1, Yusuke Oka2, Hiroya Sashida3, Toshie Kanbe4, Kenji Abe1, Iku

Utsunomiya5, and Kyoji Taguchi6

1Faculty of Pharmaceutical Sciences, Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan

Fine Pharmacy Group, Anzu Pharmacy, 877-4 Toyoshina, Azumino, Nagano 399-8205, Japan

Department of Pharmacy, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan

Departments of 4Pharmacology, 5Developmental Education and 6Medicinal Pharmacology, Showa Pharmaceutical University, 3-3165, Machida, Tokyo 194-8543, Japan

Running head: Vincristine increases TRPV1 in DRG

Address for correspondence: Kyoji Taguchi

Department of Medicinal Pharmacology, Showa Pharmaceutical University 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan Tel: +81-03-3480-5887; Fax: +81-42-721-1588 E-mail: taguchi_k@mac.com

Abstract. The clinical anti-cancer efficacy of vincristine is limited by the development of dose-dependent peripheral neuropathy. Up-regulation of transient receptor potential vanilloid 1 (TRPV1) is correlated with peripheral neuropathy following anti-cancer drug treatment. To analyze the contribution of TRPV1 to the development of vincristine-induced mechanical allodynia/hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after vincristine treatment. Mechanical allodynia/hyperalgesia was tested with von Frey filaments 14 days after intraperitoneal administration of 0.1 mg/kg vincristine in rats. TRPV1 expression in DRGs following vincristine treatment was assessed with western blot analysis and in situ hybridization histochemistry. Vincristine-induced mechanical allodynia/hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3 mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30 mg/kg, s.c.). Vincristine treatment increased the expression of TRPV1 protein in DRG neurons. In situ hybridization histochemistry revealed that most of the TRPV1 mRNA-labeled neurons in the DRG were small in size. Immunohistochemistry showed that isolectin B4-positive small DRG neurons co-expressed TRPV1 protein 14 days after treatment. These results suggest that vincristine treatment increases TRPV1 expression in small DRG neurons. TRPV1 expression may contribute to the development of vincristine-induced painful peripheral neuropathy.

Keywords: Vincristine; TRPV1; TRPV1 antagonist; Dorsal root ganglion; Peripheral neuropathic pain

Introduction

Chemotherapeutic drugs such as vincristine, paclitaxel, and oxaliplatin are widely used to treat several types of malignant tumors. Unfortunately, these anti-cancer drugs are also associated with peripheral neuropathic pain (1, 2). The paresthesia and dysesthesia induced by the vinca alkaloid vincristine occur in the early stage of vincristine treatment. Vincristine-induced peripheral neuropathy is often resistant to standard analgesics in humans. Rodent models of vincristine-induced peripheral neuropathy have been developed to elucidate these pain mechanisms (3-5). Pharmacological studies using these models have indicated that the mechanisms underlying allodynia and hyperalgesia behavior after vincristine treatment are complex (6-8).

Recently, many studies have indicated that transient receptor potential (TRP) channels are involved in paclitaxel- or oxaliplatin-induced neuropathic pain (9, 10). TRP vanilloid 1 (TRPV1) may contribute to the development of mechanical allodynia and thermal hyperalgesia after cisplatin or oxaliplatin treatment (11, 12). We reported previously that paclitaxel treatment increases TRPV1 expression in small- and medium-diameter rat dorsal root ganglion (DRG) neurons and may contribute to functional peripheral neuropathic pain (13). Also, up-regulation of TRPV1 has been observed in DRGs in a rat model of cancer pain. Up-regulation of TRPV1 in DRG neurons and anti-cancer drug-induced mechanical allodynia in a rat model of cancer pain are inhibited by TRPV1 antagonists (14). Moreover, paclitaxel-induced behavioral hypersensitivity is prevented and reversed by administration of TRPV1 antagonists (9, 13). Vincristine neurotoxicity is dose dependent and duration dependent, as continuing treatment results in a progressive increase in the clinical (15), electrophysiological

(16), and behavioral evidence of toxicity (6). Vincristine is involved in mechanically painful

neuropathy, but expression of TRPV1 in DRG neurons after vincristine treatment has not been studied. Thus, TRPV1 seems to be involved in vincristine-induced peripheral neuropathic pain (17, 18).

In the present study, using a rat model of vincristine-induced painful peripheral neuropathy, we investigated the effects of vincristine treatment on TRPV1 expression in the DRG and the associated vincristine-induced peripheral neuropathic pain.

Materials and Methods

Experimental animals

Male Wistar rats weighing 250 to 350 g were used in the present study. All rats were housed individually in automatically controlled environmental conditions, using a 12-h light-dark cycle (lights on from 08:00 to 20:00) with free access to food and water. All animals were quarantined in centralized animal facilities for at least 7 days after arrival. Each animal was used only once. Experiments were carried out according to the guidelines for animal care and use published by the National Institutes of Health and the committee of Showa Pharmaceutical University, and all efforts were made to minimize animal suffering.

Drug administration

Seventy-six animals were divided into the experimental groups by body weight using the stratified random sampling method. Vincristine (prepared with distilled water as 1.0 mg/mL vincristine sulfate) from Novopharm (Nippon Kayaku, Tokyo, Japan) was injected i.p. (0.1 mg/kg, diluted in 1 mL saline prior to injection) in two 5-day cycles with a 3-day break between cycles. Thus, a total of 10 vincristine injections were given on days 0-4 and 7-11 as previously described (19). Ruthenium red (3 mg/kg, s.c.) or capsazepine (30 mg/kg, s.c.) was administered 30 min before von Frey tests (Fig. 1).

Mechanical stimulation

Observers blinded to the experimental conditions performed mechanical behavioral testing at the same time on days 0, 7, and 14. Briefly, rats were placed in a plastic box (23 cm

x 23 cm x 12 cm) with a wire grid floor and allowed to habituate to the environment for 30 min. The sensitivity of the plantar surface of the hind paw was measured as the withdrawal response to mechanical stimulation with von Frey filaments. Filaments of varying forces (2, 4, and 8 g) were applied to the mid-plantar surface of both hind paws. Each filament was applied to each hind paw five times in ascending order of force, with each application held for 5 sec. A 1-min rest was allowed between tests on alternate hind paws and 3-4 min between subsequent tests on the same hind paw. A positive response was recorded if the paw was withdrawn during the application of the von Frey filament or immediately after its removal. Withdrawal responses to the von Frey filaments from both hind paws were counted and expressed as an overall percentage response, e.g., if a rat withdrew three out of the total 10 von Frey applications, this was recorded as a 30% overall response to the von Frey filament. The von Frey test was performed before the first vincristine administration (day 0) and on days 7 and 14 after the first dose (Fig. 1).

Western blot analysis

The rats were deeply anesthetized with pentobarbital (60 mg/kg, i.p.) on day 14 after the start of vincristine treatment. While under anesthesia, rats were euthanized by transcardial perfusion with 50 mL potassium-free phosphate-buffered saline (K+-free PBS; pH 7.4) followed by 500 mL cold 4% paraformaldehyde, and lumbar DRGs (L4-6) were rapidly dissected according to the method of Hirade et al. (20) and Malin et al. (21). DRGs (L4-6) were collected and homogenized in cold extraction buffer consisting of 10 mM Tris-HCl buffer at pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% deoxycholate.

The homogenates were centrifuged for 30 min at 15,000 xg at 4°C, and the supernatant was

collected. Total protein of the supernatant (30 mg) was electrophoresed on an SDS-polyacrylamide gel (7.5%), and separated proteins were transferred onto polyvinylidene fluoride membranes. For western blotting, anti-TRPV1 antibody (Alomone Labs, Jerusalem, Israel, 1:200) and anti-TRPV2 antibody (Alomone Labs, 1:200) were used, and anti-P-actin antibody (Sigma-Aldrich, St. Louis, MO, 1:1000) was used as the loading control. Horseradish peroxidase-labeled rabbit antibody (1:2000) was used as the secondary antibody. Specific bands were detected using enhanced chemiluminescence with the TM Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's protocol. The intensities of immunoreactive bands were analyzed with MultiGage Ver. 3 software (Fuji Film, Tokyo, Japan).

In situ hybridization histochemistry (ISHH)

Rats were perfused and DRGs were fixed and dissected for ISHH as described above in

the western blot analysis section. Following post-fixation and cryoprotection in 30% sucrose

in PBS, single DRGs were embedded in OCT, frozen at -80°C, and sectioned at a thickness

of 10 p,m. Sections were thaw-mounted onto silane-coated glass slides and fixed in 4%

paraformaldehyde in PBS for 10 min. After washing in PBS, the sections were treated with 1

|ig/mL proteinase K (Sigma-Aldrich) in PBS for 10 min at room temperature, post-fixed in

the same fixative, acetylated with acetic anhydride in 0.1 M triethanolamine, prehybridized

for 60 min at 55°C, and hybridized with digoxygenin (DIG)-labeled RNA probes overnight at

55°C. DIG-labeled sense and anti-sense RNA probes corresponded to nucleotides 865-1365

of rat TRPV1 mRNA (AY496961). Following post-hybridization washes and blocking,

sections were incubated for 120 min in anti-DIG antibody conjugated to alkaline phosphatase

(1:5000, Roche, Mannheim, Germany), and signal was visualized using nitro blue tetrazolium/bromochloroindolyl phosphate substrates (Roche). The equivalent sense probe displayed no signal.

Image analysis

Signals were analyzed with fluorescence microscopy at x400 magnification using a microscopy digital camera system. Experimenters who were unaware of the experimental protocol counted cells in a blinded manner. Neurons with visible nuclei were used for calculation. A total of five sections (90 p,m apart) were randomly selected from each DRG. The ratio of TRPV1-positive cells in the total profile was calculated for day 14 after vincristine or saline treatment. Signal intensity and area frequency analysis of each neuron were calculated with ImageJ 1.46. Neurons were considered TRPV1 positive if their signal intensity was 3-fold higher than background. The proportion of TRPV1-positive cells per total was calculated according to the size of the cell body. At least 300 neurons from each DRG of each rat were measured.

Immunohistochemistry with DRGs

On day 14 after the start of vincristine treatment, the rats were deeply anesthetized with

pentobarbital (60 mg/kg, i.p.) Rats were perfused as described above in the western blot

analysis section, and DRGs (L4-6) were removed, post-fixed for 3 h, cryoprotected overnight

in 25% sucrose solution, and stored at -80°C until use. DRG was cut at 10 p,m thickness,

thaw-mounted on silane-coated glass slides, and air-dried overnight at room temperature.

DRG sections were incubated with anti-TRPV1 antibodies (Alomone Labs, 1:200) in

blocking buffer containing 2% bovine serum albumin/0.1% Triton X-100 in K-free PBS overnight at 4°C. After rinsing, the sections were incubated in tetramethylrhodamine-5-isothiocyanate (5-TRITC)-conjugated anti-rabbit IgG (Sigma-Aldrich, 1:100) for 1 h at room temperature. The sections were then thoroughly rinsed in PBS and incubated in fluorescein isothiocyanate (FITC)-conjugated isolectin B4 (Enzo Life Science, Ontario, Canada, 1:100) for 4 h at 4°C. All immunostained DRG sections were treated with Permafluor™ (Thermo Shandon, Pittsburgh, PA), coverslipped, and evaluated with an Olympus laser-scanning confocal microscope (FLUOVIEW BW50, Olympus, Tokyo, Japan) at fluorescence wavelengths of 488 nm and 568 nm.

All sections were treated with Permafluor™ (Thermo Shandon), coverslipped, and evaluated with an Olympus microscope. Optical density of the stained sites was determined with ImageJ 1.46, an open source Java-based computer program. The optical density of TRPV1-positive cells was calculated on day 14 after vincristine or saline treatment.

Statistical analysis

All data are expressed as the mean ± SEM. In the two-group analysis including the behavioral study, the statistical significance of the difference between the saline and vincristine treatment was estimated using the F-test, followed by the Student's or Aspin-Welch's t-test. The significance of the difference in the response rate among the saline, vincristine, and ruthenium red or capsazepine groups was calculated using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer method. Statistical significance was accepted at P < 0.05.

Results

The effects of vincristine on mechanical allodynia/hyperalgesia

As expected, the mean paw withdrawal frequency was significantly increased as determined with the von Frey test 14 days after the start of vincristine treatment (0.1 mg/kg, n = 8) compared to saline (n = 8) (Fig. 2). Vincristine produced a significant increase on days 7 and 14 compared to the paw withdrawal incidences (%) for saline. On day 14, the responses to 2-g von Frey filament stimulation were significantly increased by 38.4 ± 7.0% (P < 0.01, n = 6) compared to saline treatment (13.3 ± 7.1%, Fig. 2A). Similar results were obtained in response to 4-g von Frey filament stimulation (Fig. 2B). On day 14, the responses to 8-g von Frey filament stimulation with vincristine (0.1 mg/kg) treatment were further increased compared to saline treatment (Fig. 2C). The response value was 73.9 ± 4.1%, which was significantly higher (P < 0.01) than saline treatment (52.5 ± 4.4%, Fig. 2C). Thus, vincristine-treated rats developed mechanical allodynia/hyperalgesia.

The effect of TRPV1 antagonists on vincristine-induced mechanical allodynia/hyperalgesia

We next investigated the effects of the TRP antagonist ruthenium red and TRPV1

antagonist capsazepine on vincristine-induced mechanical allodynia/hyperalgesia.

Administration of ruthenium red (3 mg/kg, s.c., n = 8) or capsazepine (30 mg/kg, s.c., n = 8)

30 min before von Frey tests significantly inhibited mechanical allodynia/hyperalgesia on day

14 after vincristine treatment (n = 8). In addition, the withdrawal incidences with

administration of ruthenium red or capsazepine alone were comparable to the saline-treated

group (2 g: 15.9 ± 4.1%, 4 g: 33.7 ± 4.9%, 8 g: 47.8 ± 7.2% in saline + ruthenium red

treatment and 2 g: 11.3 ± 7.0%, 4 g: 33.3 ± 7.2%, 8 g: 47.5 ± 8.8% in saline + capsazepine treatment, Fig. 3A, B). Thus, the TRP antagonist ruthenium red and TRPV1 antagonist capsazepine reversed vincristine-induced mechanical allodynia/hyperalgesia.

The effect of vincristine treatment on TRPV1 and TRPV2 protein expression in DRG neurons

We investigated the expression of TRPV1 and TRPV2 protein expression after vincristine treatment (0.1 mg/kg, i.p.). We removed DRGs (L4-6) at day 14 after vincristine treatment, and TRPV1 and TRPV2 protein expression was quantified with western blot analysis and compared with b-actin protein expression. As shown in Figure 4A, vincristine (0.1 mg/kg, i.p.) treatment significantly increased TRPV1 protein expression in DRGs at day 14 (40.0 ± 3.5%, n = 4, P < 0.05). In contrast, no apparent change was observed in the expression of TRPV2 protein at day 14 after vincristine treatment (Fig. 4B).

The effect of vincristine treatment on TRPV1 mRNA on ISHH

ISHH revealed that most TRPV1 mRNA-labeled neurons in the DRGs (L4-6) were small or medium in size (Fig. 5A), consistent with previous studies (13, 22). Most TRPV1 mRNA-positive DRG neurons in vincristine-treated rats were small-sized neurons compared to saline treatment (Fig. 5B). Using computerized image analysis, we found that vincristine (0.1 mg/kg) induced a significant increase in the percentage of TRPV1 mRNA-positive DRG neurons at day 14 (58.7 ± 3.5%, n = 4, P < 0.01) (Fig. 5C). Thus, vincristine increased the number of small-diameter DRG neurons that express TRPV1 mRNA, especially small-diameter DRG neurons (Fig. 5D).

The effect of vincristine treatment on TRPV1 and isolectin B4 co-expression in DRG neurons

Using immunohistochemistry, we detected TRPV1 protein expression in DRG (L4-6) neurons at day 14 after vincristine treatment. As shown in Figure 6A, double immunofluorescence experiments revealed that TRPV1 expression overlapped with isolectin B4-positive small-diameter DRG neurons (yellow in Fig. 6A). In saline-treated rats, 14.0% of the isolectin B4-positive small-diameter DRG neurons were immunostained for TRPV1. The percent of TRPV1/isolectin B4 dual-positive cells relative to the total isolectin B4-positive neurons increased at day 14 (32.8%) after vincristine treatment (Fig. 6B). Thus, vincristine treatment increased co-expression of TRPV1 and isolectin B4 in small-diameter DRG neurons at day 14.

Discussion

Peripheral neuropathic pain is one of the major side effects of chemotherapeutic drugs such as vincristine. In the present study, we evaluated TRPV1 expression in DRGs and the effect of TRP antagonists after vincristine treatment. Mechanical allodynia/hyperalgesia was observed on days 7 and 14 after the start of 0.1 mg/kg vincristine that was administered with two 5-day cycles with a 3-day break between cycles. In previous experimental studies, administration of multiple doses of vincristine produced mechanical allodynia/hyperalgesia in the von Frey test (4, 6, 23). Our results are consistent with these previously published findings. We observed that administration of the TRP antagonist ruthenium red or the TRPV1 antagonist capsazepine reversed the vincristine-induced mechanical allodynia/hyperalgesia. Similarly, antagonists of TRPV1 channels attenuate paclitaxel-induced neuropathic pain (24, 25). Therefore, we hypothesize that vincristine induces activation of TRPV1, which induces peripheral neuropathic pain. Thus, TRPV1 antagonists may inhibit vincristine-induced mechanical allodynia/hyperalgesia by blocking TRPV1 activation.

Several investigators have identified the close relationship between nerve growth factor and expression of TRPV1 (26, 27, 28). Reports have shown that administration of nerve growth factor by injection increases expression of TRPV1 in DRG neurons. Other studies of chemotherapeutic drugs, inflammation, and nerve injury have shown increased TRPV1 mRNA and protein in DRGs (22, 29, 30). Moreover, vincristine treatment increases the activity of TRPV4 in the sensory neurons of the DRG (31). In the present study, we found that vincristine-treated rats developed mechanical allodynia/hyperalgesia and increased levels of TRPV1 protein expression in the DRG. In contrast, TRPV2 protein expression did not change in DRGs after vincristine treatment. Thus, vincristine-induced neuropathy is likely mediated

by TRPV1 but not TRPV2. We speculate that vincristine-induced neuropathic pain may be the result of up-regulation of TRPV1 protein expression in DRG neurons. In addition, we first demonstrated that vincristine treatment significantly increased the expression of TRPV1 protein in the rat DRG, and ISHH revealed that this TRPV1 mRNA expression was increased in small-diameter DRG neurons following vincristine treatment. Moreover, DRG sections were double-labeled for TRPV1 and the small-diameter DRG neuron marker, isolectin B4. The ratio of neurons expressing TRPV1 among those that were isolectin B4-positive was significantly higher in vincristine-treated rats, confirming that a considerable number of C-fiber neurons began to express TRPV1 after vincristine treatment. Breese and colleagues (32) showed that isolectin B4-positive small-diameter DRG neurons show increased TRPV1 function and expression after peripheral inflammation. Vincristine may have increased the expression of TRPV1 in small-diameter DRG neurons, and the up-regulation of TRPV1 may be due to the onset of vincristine-induced mechanical allodynia/hyperalgesia hyperalgesia. Therefore, TRPV1 likely plays an important role in vincristine-induced peripheral neuropathic pain.

Regarding peripheral neuropathic pain from chemotherapeutic drugs, substance P in the

spinal cord dorsal horn may contribute to the development of mechanical allodynia and

thermal hyperalgesia after paclitaxel treatment (17, 18). Our group demonstrated that

paclitaxel treatment increases the release of substance P in the superficial layers of the spinal

dorsal horn (17). In electrophysiological studies, hypersensitivity of C-fiber nociceptors in the

spinal cord has been shown during vincristine-induced neuropathic pain (33). In addition,

central sensitization of wide dynamic range neurons in the spinal dorsal horn has also been

observed following vincristine treatment (34). Further studies are necessary to define the role

of vincristine in substance P release in the spinal cord via activation of TRPV1.

In conclusion, these observations suggest that vincristine-induced painful peripheral neuropathy is caused by up-regulation of TRPV1 in small-diameter DRG neurons.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Figure Legends

Fig. 1. Experimental time schedule of administration of vincristine using the von Frey test. Baseline withdrawal responses were measured on day 0 before saline or vincristine (0.1 mg/kg, i.p.) treatment. Either vincristine (0.1 mg/kg) or saline was injected intraperitoneally in two 5-day cycles with a 3-day break between cycles. Mechanical withdrawal response was measured on days 7 and 14 after saline or vincristine treatment. Ruthenium red (3 mg/kg, s.c.) or capsazepine (30 mg/kg, s.c.) was administered 30 min before von Frey tests on day 14.

Fig. 2. Behavioral responses of mechanical allodynia/hyperalgesia induced by vincristine treatment (0.1 mg/kg, i.p.). The von Frey test was used to measure mechanical allodynia/hyperalgesia induced by vincristine treatment. Histograms show the mean ± SEM (n = 8 for each group) of withdrawal response (frequency following mechanical stimulation) at days 0, 7, and 14 after vincristine treatment or saline using a 2-g (A), 4-g (B), or 8-g (C) von Frey filament (vFF). Symbols indicate a significant difference compared to the saline. *P < 0.05, **P < 0.01. The significance of the difference was analyzed using the F-test, followed by the Student's or Aspin-Welch's t-test, compared to the saline treatment group.

Fig. 3. Effect of ruthenium red (RR) and capsazepine (CPZ) on the von Frey tests at day 14 after vincristine (0.1 mg/kg, i.p.) treatment. Histograms show the mean ± SEM (n = 8-10 for each group) of the response frequency to mechanical stimulation by 2-g, 4-g, and 8-g von Frey filaments (vFF). RR (3 mg/kg, s.c.) or CPZ (30 mg/kg, s.c.) was administered 30 min before von Frey tests. *P < 0.05, **P < 0.01 compared to saline, #P < 0.05 compared to

vincristine alone. The significance of the difference in the withdrawal frequency among the groups was calculated using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer method.

Fig. 4. Effects of vincristine treatment on expression of TRPV1 and TRPV2 protein in rat DRGs (L4-6). A, B, Expression of TRPV1 (A) or TPRV2 (B) protein in DRGs at day 14 after saline and vincristine treatment (0.1 mg/kg) was measured. TRPV1 and TRPV2 protein expression was normalized to ß-actin expression. Histograms show the relative amount of TRPV1 or TRPV2 protein in vincristine-treated rats compared with saline-treated rats. The western blot shows representative data. Data are the mean ± SEM. n = 4 each for vincristine treatment and saline. *P < 0.05 versus saline.

Fig. 5. Effect of vincristine treatment on TRPV1 mRNA expression in rat DRG neurons. A and B, Representative photomicrographs of TRPV1 mRNA in situ hybridization histochemistry on day 14 after saline and vincristine treatment (0.1 mg/kg, i.p.). DRG (L4-6) sections were incubated with anti-TRPV1 DIG-labeled probes. C, Optical density of TRPV1 mRNA-stained DRG neurons 14 days after the start of saline or vincristine treatment. A total of about 1000 DRG neurons each from saline-treated rats and vincristine-treated rats were measured. D, Size distribution of TRPV1 mRNA-expressing neurons in DRGs in saline- and vincristine-treated rats. TRPV1 mRNA expression was distributed among all sizes of neurons, but most TRPV1 mRNA expression was found in small DRG neurons. Data are the mean ± SEM. n = 4 each for saline treatment and vincristine treatment. **P < 0.01 versus saline.

Fig. 6. Effect of vincristine treatment on TRPV1 protein expression in rat DRG neurons. A, Photomicrographs showing immunohistochemistry for TRPV1 protein at day 14 after vincristine treatment (0.1 mg/kg, i.p.). TRPV1 protein-expressing neurons were more frequently observed after vincristine treatment. The photograph shows representative data. Co-localization of TRPV1 (red) and isolectin B4 (green). Double-labeled neurons (arrowheads) are stained yellow in the merged panel. B, Pie chart shows the percent of TRPV1-positive neurons relative to the total neurons. TRPV1 protein-expressing neurons were more frequently observed at day 14 after vincristine (0.1 mg/kg, i.p.) treatment. n = 4 each for saline and vincristine treatment.

Vincristine (0.1 mg/kg) Vincristine (0.1 mg/kg) Mechanical threshold

treatment treatment testing

rTTTi iTiïl ♦

LJ_I_I_I_I_I—I_I_I_I_I_I_I_I

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (days)

Mechanical threshold Mechanical threshold Capsazepine or

testing testing Ruthenium red

(Before administration) (Before administration) (30 min before testing)

□ Saline H Vincristine (0.1 mg/kg)

A: vFF 2 g

B:vFF 4 g

C: vFF 8 g

day 0 day 7 day 14

day 0 day 7 day 14

day 0 day 7 day 14

A: Effect of RR

□ Saline ■ saline + RR (3 mg/kg) H Vincristine (0.1 mg/kg) ^Vincristine + RR (3 mg/kg)

B: Effect of CPZ

□ Saline ■ saline + CPZ (30 mg/kg) H Vincristine (0.1 mg/kg) IÜ Vincristine + CPZ (30 mg/kg)

vFF 2 g vFF 4 g vFF 8 g

vFF 2 g vFF 4 g vFF 8 g

Fig. 4

A: TRPV1

B: TRPV2

TRPVl ß-actin

c > p«

Saline Vincristine (0.1 mg/kg)

Saline

Vincristine (0.1 mg/kg)

A: Saline

B: Vincristine

100 lim

D: Neuronal profile

120 100 80 60 40 20 0

C: TRPV1 density 200

£ 100

Saline Vincristine (0.1 mg/kg)

I iü-iTTTü-ii

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

Neuronal diameter (/im2)

Fig. 6 A

Saline

Isolectin B4

merged

B co-localized: 14.0%

TRPV1 only: 86.0%

Vincristine (0.1 mg/kg)

- • f:

X f 4 " M

co-localized: 32.9%

TRPV1 only: 67.1%

100 /im