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Paclitaxel-induced peripheral neuropathy increases substance P release in rat spinal cord
European Journal of Pharmacology 770 (2016) 46–51
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Paclitaxel-induced peripheral neuropathy increases substance P release in rat spinal cord Terumasa Chiba a, Yusuke Oka a, Toshie Kambe b, Naoya Koizumi c, Kenji Abe d, Kazuyoshi Kawakami e, Iku Utsunomiya f, Kyoji Taguchi a,n a
Departments of Medicinal Pharmacology, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan Departments of Pharmacology, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan c Departments of Pharmaceutics and Biopharmaceutics Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan d Department of Pharmacology, School of Pharmaceutical Sciences, Ohu University, 31-1 Tomitamachi, Koriyama, Fukushima 963-8611, Japan e Department of Pharmacy, Cancer Institute Hospital, 3-10-6 Ariake, Koto-Ku, Tokyo 135-8550, Japan f Departments of Developmental Education, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 May 2015 Received in revised form 26 November 2015 Accepted 27 November 2015 Available online 30 November 2015
Peripheral neuropathy is a common adverse effect of paclitaxel treatment. The major dose-limiting side effect of paclitaxel is peripheral sensory neuropathy, which is characterized by painful paresthesia of the hands and feet. To analyze the contribution of substance P to the development of paclitaxel-induced mechanical hyperalgesia, substance P expression in the superficial layers of the rat spinal dorsal horn was analyzed after paclitaxel treatment. Behavioral assessment using the von Frey test and the paw thermal test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced mechanical allodynia/hyperalgesia and thermal hyperalgesia 7 and 14 days after treatment. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment significantly increased substance P expression (37.6 73.7% on day 7, 43.6 7 4.6% on day 14) in the superficial layers of the spinal dorsal horn, whereas calcitonin gene-related peptide (CGRP) expression was unchanged. Moreover, paclitaxel (2 and 4 mg/kg) treatment significantly increased substance P release in the spinal cord on day 14. These results suggest that paclitaxel treatment increases release of substance P, but not CGRP in the superficial layers of the spinal dorsal horn and may contribute to paclitaxel-induced painful peripheral neuropathy. & 2015 Elsevier B.V. All rights reserved.
Keywords: Paclitaxel Substance P Calcitonin gene-related peptide Spinal cord Peripheral neuropathic pain
1. Introduction Paclitaxel is commonly used to breast and ovarian cancer and non-small cell lung carcinoma by promoting microtubule dysfunction (Wilson and Jordan, 2004). Anticancer treatments such as paclitaxel induce peripheral neuropathy, which is the main reason for either changing or stopping chemotherapy. Rodent models of paclitaxel-induced neuropathy have been developed to elucidate these pain mechanisms (Polomano et al., 2001; Dougherty et al., 2004; Cata et al., 2006; Tatsushima et al., 2011a). Recently, we reported that paclitaxel treatment up-regulates transient receptor potential vanilloid 1 (TRPV1) in small- and medium-diameter rat dorsal root ganglion (DRG) neurons and that paclitaxel-induced neuropathy is prevented by pretreatment with TRPV1 antagonists (Hara et al., 2013). Pharmacological studies using these models have indicated that the mechanisms underlying mechanical and n
Corresponding author. E-mail address: [email protected] (K. Taguchi).
http://dx.doi.org/10.1016/j.ejphar.2015.11.055 0014-2999/& 2015 Elsevier B.V. All rights reserved.
thermal hyperalgesia behavior after paclitaxel treatment are complex (Itoh et al., 2004a; Chen et al., 2011; Xiao et al., 2011; Kawakami et al., 2012). Substance P, an important neurotransmitter in primary sensory neurons, plays an important role in a number of sensory, autonomic, and cognitive functions, primarily by binding to the neurokinin-1 (NK1) receptor. Primary sensory neurons are specialized to transmit sensory information from the periphery to the spinal cord. Previous studies have suggested that substance P- and calcitonin gene-related peptide (CGRP)-immunopositive nerve terminals play an important role in the regulation of peripheral signals (Ambalavanar, et al., 2003; Joachim et al., 2007). Substance P has long been proposed to contribute to nociceptive signaling within the spinal cord dorsal horn, where it is released by nociceptive primary afferent fibers (Morris et al., 2004). Paclitaxel increases substance P and CGRP in plasma and bronchoalveolar lavage fluid (Itoh et al., 2004b). Substance P binds NK1 and NK2 receptors (Regoli et al., 1989), and coadministration of NK1 and NK2 antagonists reverses paclitaxel-induced peripheral neuropathy (Tatsushima et al., 2011b). Moreover, paclitaxel
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evokes the release of substance P from cultured DRG cells, and pemirolast, an antiallergic agent, inhibits paclitaxel-induced substance P release (Tatsushima et al., 2011b). However, the role of substance P and CGRP in the spinal cord in paclitaxel-induced peripheral neuropathic pain remains unknown. In the present study, using a rat model of paclitaxel-induced, painful peripheral neuropathy, we investigated the effects of paclitaxel treatment on substance P and CGRP in the spinal cord.
2. Materials and methods 2.1. Experimental animals Male Wistar rats weighing 250–300 g (Japan Laboratory Animals, Inc., Tokyo, Japan) 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. 2.2. Drug administration Paclitaxel (2 or 4 mg/kg/ml; Taxol; Bristol-Myers-Squibb; 6 mg/ ml paclitaxel in Cremophor EL and dehydrated ethanol) was diluted with saline. Vehicle was prepared from 1 part vehicle stock solution and 2 parts saline (vehicle stock solution: Cremophor ELpolyethoxylated castor oil and ethanol at a 1:1 ratio). Paclitaxel (cumulative dose, 8 or 16 mg/kg) or vehicle was injected intraperitoneally (i.p.) on four alternate days (days 0, 2, 4, and 6) as previously described (Hara et al., 2013). According to the prescribing information sheet for the administration of paclitaxel, the recommended dose is 135–175 mg/m2 (TAXOL package insert, Bristol-Myers Squibb Company, 2003), which is converted into approximately 3.6–4.7 mg/kg (height, 1.7 m; weight, 65 kg; Du Bois formula). In previous experimental studies, paclitaxel has been administered multiple times on consecutive or alternate days or a single dose has been given (2 and 4 mg/kg) (Polomano et al., 2001; Flatters and Bennett, 2004; Smith et al., 2004; Matsumoto et al., 2006). 2.3. Mechanical stimulation Observers blinded to the experimental conditions performed mechanical behavioral testing at the same time on days 0, 7, and 14. In brief, rats were placed in a plastic box with a wire grid floor and allowed to habituate to the environment for 20 min. The sensitivity of the plantar surface of the hind paw was measured as the withdrawal responses to mechanical stimulation with von Frey filaments. Filaments of varying forces (2.0 and 5.0 g) were applied to the mid-plantar surface of both hind paws. In ascending order of force, each filament was applied to each hind paw five times, with each application held for 5 s. 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 paclitaxel administration (day 0)
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and on days 7 and 14 after the first dose. 2.4. Thermal stimulation Observers blinded to the experimental conditions tested the rats with thermal stimulation. The thermal stimulation test was performed before the first paclitaxel administration (day 0) and on days 7 and 14 after the first dose. In brief, rats were placed in a plastic box on a glass surface and allowed to habituate to the environment for 20 min. We used a radiant thermal stimulator (IITC Inc., Woodland Hills, CA), and the intensity of the light was adjusted at the start of the experiment so that average baseline latencies were approximately 9 s with a cut-off latency of 20 s. The focus of radiant thermal stimulation was aimed exactly on the middle of the plantar surface of each hind paw, and the latency to paw withdrawal was recorded. Three trials were performed at intervals of 5 min, and one score was assigned for each session by averaging the last two trials. The latencies were obtained alternately from the left and right hind paws, which were tested 5 min apart. The data were reported as the mean values for both the right and left hind paws combined. 2.5. Immunohistochemistry The rats were deeply anesthetized with pentobarbital (50 mg/ kg, i.p.) on day 7 or 14 after the start of paclitaxel (2 or 4 mg/kg) treatment, and the spinal cord was prepared for immunohistochemistry. Rats were perfused transcardially with 20 ml potassium-free phosphate-buffered saline (K þ -free PBS; pH 7.4) followed by 50 ml 4% paraformaldehyde solution. The spinal cord (L4–6) was removed, post-fixed for 3 h, cryoprotected overnight in 25% sucrose solution, and then stored at 80 °C until use. Spinal cords were cut at 15-μm thickness, thaw-mounted on silanecoated glass slides, and air-dried overnight at room temperature. Spinal cord sections were incubated with excess blocking buffer containing 2% skim milk in 0.1% Triton X-100 in K þ -free PBS and subsequently reacted overnight at 4 °C with the substance P Immunohistochemistry Staining Kit (Peninsula Laboratories LLC, San Carlos, CA) and rabbit polyclonal antiserum to CGRP (Enzo, Farmingdale, NY). The sections were then incubated in fluorescein isothiocyanate-conjugated anti-rabbit IgG (Sigma-Aldrich, Inc., St. Louis, MO, 1:200) for 2 h at room temperature. All sections were treated with Permafluor™ (Thermo Shandon, Pittsburgh, PA), coverslipped, and evaluated with microscopy. The immunostained sections were mounted on slides, covered with coverslips, and observed with an Olympus microscope (Olympus, Tokyo, Japan). The optical density (OD) of the stained sites was determined with ImageJ 1.46, an open source Java-based computer program. The OD of substance P and CGRP were calculated for days 7 and 14 after the start of paclitaxel or cremophor vehicle treatment. 2.6. Image analysis Signals were analyzed with fluorescence microscopy at 400 magnification using a microscopy digital camera system. Experimenters who were unaware of the experimental protocol observed the spinal cord sections. Five sections were randomly selected from each spinal cord (L4–6). The ratio of the area of substance P immunoreactivity to the area of CGRP immunoreactivity in the total profile was calculated for days 7 and 14 after the start of paclitaxel or vehicle treatment. Signal intensity and area frequency analysis of each neuron were calculated with ImageJ 1.46.
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2.7. Determination of substance P levels Immediately after sacrifice, the lumbar spinal cord was rapidly dissected out and frozen at 80 °C for substance P analysis. The lumbar spinal cord samples were cut into approximately 10-mm cubes and homogenized in a cold extraction buffer consisting of 20 mM Tris–HCl buffer at pH 8.0, with 100 mM Trizmas base, 400 mM NaCl, 4 mM EDTA, 1 mM PMSF, 7 mg/ml aprotinin, and 10% Triton X-100. The homogenates were vortexed for 2 min, centrifuged for 90 min at 42,000 g, and the supernatants were collected. The levels of substance P in the homogenates were measured with a competitive enzyme immunoassay (Substance P EIA kit; Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. The substance P EIA kit is a competitive assay that provides accurate measurement of substance P with a working range of 3.9–500 pg/ml, with a typical IC50 of approximately 30 pg/ml. 2.8. Statistical analysis All data are expressed as the mean 7S.E.M. The F-test followed by an unpaired t-test (Student's or Aspin-Welch's t-test) or oneway analysis of variance (ANOVA) followed by Dunnett's multiple comparison test were used to compare the effects of each paclitaxel dose and vehicle treatment. Statistical significance was established at P o0.05.
3. Results 3.1. The effects of paclitaxel on mechanical allodynia/hyperalgesia As expected, the mean paw withdrawal reflex was significantly increased with both 2 and 4 mg/kg paclitaxel treatment as determined with the von Frey test 7 and 14 days after the start of paclitaxel treatment compared to the cremophor vehicle (Fig. 1). On day 7, the responses to 2-g von Frey filament stimulation were significantly increased by 43.9 79.8% (2 mg/kg, P o0.01, n ¼ 6) and 51.77 12.4% (4 mg/kg, P o0.01, n ¼ 6) compared to vehicle treatment (9.57 3.6%, Fig. 1A). Paclitaxel also produced a significant increase on day 14, when the paw withdrawal incidences (%) for vehicle, 2, and 4 mg/kg paclitaxel treatment were 12.5 7 4.1%, 62.8 710.0% (P o0.01), and 66.2 712.7% (P o0.01), respectively. Similar results were obtained in response to 5-g von Frey filament stimulation. On day 7, withdrawal incidences for 2 mg/kg (58.7 77.6%) and 4 mg/kg treatment (68.37 7.2%) were both significantly increased compared to vehicle control (25.8 73.7%). On day 14, the responses to 5-g von Frey filament stimulation with paclitaxel (2 and 4 mg/kg) treatment were further increased compared to cremophor vehicle treatment. The response values were 91.1 77.8% (2 mg/kg) and 95.1 78.2% (4 mg/kg), and these were both significantly higher (P o0.01) than cremophor vehicle treatment (22.6 7 4.0%, Fig. 1B). 3.2. The effect of paclitaxel on thermal hyperalgesia We next investigated the effect of paclitaxel (2 and 4 mg/kg, i. p.) on thermal hyperalgesia. On day 7, the latency withdrawal time for thermal stimulation following cremophor vehicle treatment (7.83 70.50 s) was significantly decreased to 6.58 70.46 s (P o0.05, n ¼5) with paclitaxel (4 mg/kg) treatment. On day 14, the latency withdrawal time for thermal stimulation following paclitaxel (4 mg/kg: 5.747 0.33 s) treatment was further
Fig. 1. Effect of paclitaxel-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia as determined with the von Frey test and paw thermal test, respectively. (A, B) von Frey filaments (2 g and 5 g) were used to measure mechanical allodynia/hyperalgesia induced by paclitaxel treatment (2 and 4 mg/kg, i.p.; administered on days 0, 2, 4, and 6) in rats. Histograms show the mean 7 S.E.M. of the withdrawal response on days 7 and 14 after the start of paclitaxel or cremophor vehicle treatment. (C) The paw thermal test was used to measure thermal hyperalgesia induced by paclitaxel treatment (2 and 4 mg/kg, i.p.; administered on days 0, 2, 4, and 6) in rats. Histograms show the mean 7S.E.M. of the withdrawal response at days 7 and 14 after the start of paclitaxel and cremophor vehicle treatment. Data are the mean 7 S.E.M. of n ¼5–6 rats. *Po 0.05, **Po 0.01, one-way ANOVA with Dunnett’s post-hoc analysis compared to cremophor vehicle treatment.
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Fig. 2. Effects of paclitaxel treatment on substance P expression in the rat spinal cord. (A) Photomicrographs showing immunoreactivity for substance P in the lumbar dorsal horn of the spinal cord 14 days after the start of cremophor vehicle or paclitaxel (4 mg/kg) treatment. Scale bar, 100 μm. (B) OD of substance P immunostaining on days 7 and 14 after the start of cremophor vehicle or paclitaxel treatment. Histograms show the substance P OD in paclitaxel-treated (2 and 4 mg/kg) rats relative to cremophor vehicletreated rats. The blue box is the region used to obtain the OD. Data are the mean 7 S.E.M. n¼ 5 each for paclitaxel and cremophor vehicle treatment. One-way ANOVA with Dunnett’s post-hoc analysis compared to cremophor vehicle treatment. **Po 0.01 versus cremophor vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
decreased compared to cremophor vehicle treatment (7.55 70.30 s, P o0.01, n ¼ 5). Paclitaxel (2 mg/kg) treatment also significantly decreased the latency withdrawal time for thermal stimulation at 14 days (5.92 7 0.42 s, Po 0.01, n ¼5, Fig. 1C). 3.3. The effect of paclitaxel treatment on substance P expression and CGRP expression in superficial layers of the spinal dorsal horn Using immunohistochemistry, we detected substance P protein expression in the rat superficial layers of the spinal dorsal horn (layers I–II) at days 7 and 14 after the start of paclitaxel treatment. Compared with cremophor vehicle-treated rats, substance P protein expression was more intense after paclitaxel treatment (Fig. 2A). Using computerized OD image analysis, we measured the OD of superficial layers of the spinal dorsal horn that showed substance P protein expression. Paclitaxel (2 and 4 mg/kg) treatment induced a significant increase in the substance P OD in superficial layers of the spinal dorsal horn at day 14 (2 mg/kg: 24.07 2.4%; 4 mg/kg: 43.6 7 4.6%, P o0.01, n ¼5) compared with substance P protein expression in cremophor vehicle-treated rats (Fig. 2B). On the other hand, CGRP expression in the superficial layers of the spinal dorsal horn did not increase at day 14 following paclitaxel (4 mg/kg, n ¼5) treatment (Fig. 3A). Also, paclitaxel (4 mg/kg) treatment did not affect the CGRP OD in superficial layers of the spinal dorsal horn at days 7 and 14 (Fig. 3B). Thus,
substance P but not CGRP was significantly increased in the superficial layers of the spinal dorsal horn (layers I–II) in paclitaxeltreated rats. 3.4. The effect of paclitaxel treatment on substance P release in the spinal cord We removed the spinal cord at 14 days after the start of 2 and 4 mg/kg paclitaxel treatment. Release of substance P was quantified with a competitive enzyme immunoassay and compared with cremophor vehicle treatment. Substance P release was significantly increased both 2 mg/kg (700.0 7 67.8 pg/mg, P o0.05, n ¼5) and 4 mg/kg (959.3 725.4 pg/mg, P o0.01, n ¼ 4) paclitaxel treatment compared to cremophor vehicle treatment (524.3 780.9 pg/mg, Fig. 4). Thus, paclitaxel (2 and 4 mg/kg) treatment significantly increased the release of substance P in the spinal cord on day 14.
4. Discussion In the present study, mechanical allodynia/hyperalgesia and thermal hyperalgesia were observed on days 7 and 14 after the start of 2 or 4 mg/kg paclitaxel that was administered on 4 alternate days (from day 0 to day 6). When comparing mechanical
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Fig. 3. Effects of paclitaxel treatment on CGRP expression in the rat spinal cord. (A) Photomicrographs of CGRP in the lumbar dorsal horn of the spinal cord 14 days after the start of cremophor vehicle or paclitaxel (4 mg/kg) treatment. Scale bar, 100 μm. (B) OD of CGRP immunostaining on days 7 and 14 after the start of cremophor vehicle or paclitaxel treatment. Histograms show the CGRP OD in paclitaxel-treated (4 mg/kg) rats relative to cremophor vehicle-treated rats. The blue box is the region used to obtain the OD. Data are the mean 7 S.E.M. n¼ 5 each for paclitaxel and cremophor vehicle treatment. The F-test followed by the Student’s or Aspin-Welch’s t-test was used for evaluation of significance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Effect of paclitaxel treatment on the substance P level in the spinal cord. The histogram shows the level of substance P in the rat spinal cord 14 days after the start of paclitaxel (2 and 4 mg/kg, i.p.) treatment. Data are the mean 7 S.E.M. n ¼4– 5 each for cremophor vehicle treatment and paclitaxel treatment. One-way ANOVA with Dunnett’s post-hoc analysis compared to cremophor vehicle treatment. *Po 0.05, **P o0.01 versus cremophor vehicle.
allodynia/hyperalgesia and thermal hyperalgesia after administration of a single i.p., multiple i.p., or single i.v. dose of paclitaxel, no significantly different result was observed between the number of doses or the route at 7 days to 28 days after paclitaxel treatment (Itoh et al., 2004b; Polomano et al., 2001; Flatters and Bennett, 2004; Matsumoto et al., 2006). In our previous experimental studies, multiple doses of paclitaxel also produced mechanical allodynia/hyperalgesia and thermal hyperalgesia (Kawakami et al., 2012; Hara et al., 2013). Thus, the results of the present study are consistent with these previously published findings. Substance P and CGRP are excitatory neurotransmitters or neuromodulators that are released in the spinal cord dorsal horn by primary sensory afferents, thus contributing to the development of allodynia and hyperalgesia by facilitating the release of excitatory neurotransmitters from primary afferents (Ma and Eisenach, 2003). Moreover, Cata et al. (2006) have shown increased levels of excitability in spinal cord dorsal horn neurons in rats with paclitaxel-evoked neuropathic pain. Vachon et al. (2004) suggested a correlation between an increase in substance P and the development of mechanical allodynia induced by sciatic nerve cuff implantation. Our immunohistochemical analysis showed that paclitaxel-treated rats showed increased expression of substance P protein in the superficial layers of the spinal cord, in addition to development of mechanical allodynia/hyperalgesia. In contrast, CGRP was not changed in the spinal cord after paclitaxel treatment. Substance P is localized in high-threshold nociceptive c-fibers and is released by noxious stimuli (Otsuka and Yoshioka, 1993). Thus, paclitaxel-induced neuropathy is likely mediated by substance P, but not CGRP. We speculate that paclitaxel-induced neuropathic pain may be the result of up-regulation of substance P protein expression in the spinal cord. Also, paclitaxel increases the release of substance P from cultured adult rat DRG neurons
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(Tatsushima et al., 2011b). Regarding the mechanism, paclitaxel evokes the release of substance P from cultured DRG cells via an extracellular Ca2 þ influx through TRP channels (Tang et al., 2006; Tang et al., 2008; Miyano et al., 2009). In the present study, cross-reactivities of 97%, 93%, and 30% for the substance P fragments SP (4–11), SP (2–11), and SP (7–11), respectively, were observed with the substance P EIA kit. Moreover, cross-reactivities for substance P fragments SP (8–11) and SP (1–4) were less than 0.01%. Cross-reactivities were 12%, 2.7%, and 0.04% for Eledosin, neurokinin A, and neurokinin B, respectively. Several studies have demonstrated that substance P can be correctly analyzed using this substance P EIA kit (Itoh et al., 2004a, 2004b; Ursavas et al., 2007; Tatsushima et al., 2011b; Oz et al., 2011). Thus, we used this analysis method for studying paclitaxelinduced substance P release in the spinal cord and demonstrated that paclitaxel treatment significantly increased the release of substance P in the rat spinal cord. In addition, intrathecal injection of NK1 and NK2 receptor antagonists strongly reverses paclitaxelinduced neuropathy (Tatsushima et al., 2011b), and NK1 receptor antagonists relieve peripheral nerve injury-induced mechanical allodynia/hyperalgesia (Cahill and Coderre, 2002). We postulate that paclitaxel-induced substance P release in the DRG may facilitate substance P transport along central nerve terminals, resulting in an increase in substance P release in the dorsal horn of the spinal cord. These findings suggest that substance P released from the spinal cord is involved in paclitaxel-induced neuropathy. In conclusion, we suggest that paclitaxel-induced neuropathic pain may be the result of the release of substance P in the spinal cord dorsal horn.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Paclitaxel-induced peripheral neuropathy increases substance P release in rat spinal cord Terumasa Chiba a, Yusuke Oka a, Toshie Kambe b, Naoya Koizumi c, Kenji Abe d, Kazuyoshi Kawakami e, Iku Utsunomiya f, Kyoji Taguchi a,n a
Departments of Medicinal Pharmacology, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan Departments of Pharmacology, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan c Departments of Pharmaceutics and Biopharmaceutics Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan d Department of Pharmacology, School of Pharmaceutical Sciences, Ohu University, 31-1 Tomitamachi, Koriyama, Fukushima 963-8611, Japan e Department of Pharmacy, Cancer Institute Hospital, 3-10-6 Ariake, Koto-Ku, Tokyo 135-8550, Japan f Departments of Developmental Education, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 May 2015 Received in revised form 26 November 2015 Accepted 27 November 2015 Available online 30 November 2015
Peripheral neuropathy is a common adverse effect of paclitaxel treatment. The major dose-limiting side effect of paclitaxel is peripheral sensory neuropathy, which is characterized by painful paresthesia of the hands and feet. To analyze the contribution of substance P to the development of paclitaxel-induced mechanical hyperalgesia, substance P expression in the superficial layers of the rat spinal dorsal horn was analyzed after paclitaxel treatment. Behavioral assessment using the von Frey test and the paw thermal test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced mechanical allodynia/hyperalgesia and thermal hyperalgesia 7 and 14 days after treatment. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment significantly increased substance P expression (37.6 73.7% on day 7, 43.6 7 4.6% on day 14) in the superficial layers of the spinal dorsal horn, whereas calcitonin gene-related peptide (CGRP) expression was unchanged. Moreover, paclitaxel (2 and 4 mg/kg) treatment significantly increased substance P release in the spinal cord on day 14. These results suggest that paclitaxel treatment increases release of substance P, but not CGRP in the superficial layers of the spinal dorsal horn and may contribute to paclitaxel-induced painful peripheral neuropathy. & 2015 Elsevier B.V. All rights reserved.
Keywords: Paclitaxel Substance P Calcitonin gene-related peptide Spinal cord Peripheral neuropathic pain
1. Introduction Paclitaxel is commonly used to breast and ovarian cancer and non-small cell lung carcinoma by promoting microtubule dysfunction (Wilson and Jordan, 2004). Anticancer treatments such as paclitaxel induce peripheral neuropathy, which is the main reason for either changing or stopping chemotherapy. Rodent models of paclitaxel-induced neuropathy have been developed to elucidate these pain mechanisms (Polomano et al., 2001; Dougherty et al., 2004; Cata et al., 2006; Tatsushima et al., 2011a). Recently, we reported that paclitaxel treatment up-regulates transient receptor potential vanilloid 1 (TRPV1) in small- and medium-diameter rat dorsal root ganglion (DRG) neurons and that paclitaxel-induced neuropathy is prevented by pretreatment with TRPV1 antagonists (Hara et al., 2013). Pharmacological studies using these models have indicated that the mechanisms underlying mechanical and n
Corresponding author. E-mail address: [email protected] (K. Taguchi).
http://dx.doi.org/10.1016/j.ejphar.2015.11.055 0014-2999/& 2015 Elsevier B.V. All rights reserved.
thermal hyperalgesia behavior after paclitaxel treatment are complex (Itoh et al., 2004a; Chen et al., 2011; Xiao et al., 2011; Kawakami et al., 2012). Substance P, an important neurotransmitter in primary sensory neurons, plays an important role in a number of sensory, autonomic, and cognitive functions, primarily by binding to the neurokinin-1 (NK1) receptor. Primary sensory neurons are specialized to transmit sensory information from the periphery to the spinal cord. Previous studies have suggested that substance P- and calcitonin gene-related peptide (CGRP)-immunopositive nerve terminals play an important role in the regulation of peripheral signals (Ambalavanar, et al., 2003; Joachim et al., 2007). Substance P has long been proposed to contribute to nociceptive signaling within the spinal cord dorsal horn, where it is released by nociceptive primary afferent fibers (Morris et al., 2004). Paclitaxel increases substance P and CGRP in plasma and bronchoalveolar lavage fluid (Itoh et al., 2004b). Substance P binds NK1 and NK2 receptors (Regoli et al., 1989), and coadministration of NK1 and NK2 antagonists reverses paclitaxel-induced peripheral neuropathy (Tatsushima et al., 2011b). Moreover, paclitaxel
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evokes the release of substance P from cultured DRG cells, and pemirolast, an antiallergic agent, inhibits paclitaxel-induced substance P release (Tatsushima et al., 2011b). However, the role of substance P and CGRP in the spinal cord in paclitaxel-induced peripheral neuropathic pain remains unknown. In the present study, using a rat model of paclitaxel-induced, painful peripheral neuropathy, we investigated the effects of paclitaxel treatment on substance P and CGRP in the spinal cord.
2. Materials and methods 2.1. Experimental animals Male Wistar rats weighing 250–300 g (Japan Laboratory Animals, Inc., Tokyo, Japan) 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. 2.2. Drug administration Paclitaxel (2 or 4 mg/kg/ml; Taxol; Bristol-Myers-Squibb; 6 mg/ ml paclitaxel in Cremophor EL and dehydrated ethanol) was diluted with saline. Vehicle was prepared from 1 part vehicle stock solution and 2 parts saline (vehicle stock solution: Cremophor ELpolyethoxylated castor oil and ethanol at a 1:1 ratio). Paclitaxel (cumulative dose, 8 or 16 mg/kg) or vehicle was injected intraperitoneally (i.p.) on four alternate days (days 0, 2, 4, and 6) as previously described (Hara et al., 2013). According to the prescribing information sheet for the administration of paclitaxel, the recommended dose is 135–175 mg/m2 (TAXOL package insert, Bristol-Myers Squibb Company, 2003), which is converted into approximately 3.6–4.7 mg/kg (height, 1.7 m; weight, 65 kg; Du Bois formula). In previous experimental studies, paclitaxel has been administered multiple times on consecutive or alternate days or a single dose has been given (2 and 4 mg/kg) (Polomano et al., 2001; Flatters and Bennett, 2004; Smith et al., 2004; Matsumoto et al., 2006). 2.3. Mechanical stimulation Observers blinded to the experimental conditions performed mechanical behavioral testing at the same time on days 0, 7, and 14. In brief, rats were placed in a plastic box with a wire grid floor and allowed to habituate to the environment for 20 min. The sensitivity of the plantar surface of the hind paw was measured as the withdrawal responses to mechanical stimulation with von Frey filaments. Filaments of varying forces (2.0 and 5.0 g) were applied to the mid-plantar surface of both hind paws. In ascending order of force, each filament was applied to each hind paw five times, with each application held for 5 s. 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 paclitaxel administration (day 0)
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and on days 7 and 14 after the first dose. 2.4. Thermal stimulation Observers blinded to the experimental conditions tested the rats with thermal stimulation. The thermal stimulation test was performed before the first paclitaxel administration (day 0) and on days 7 and 14 after the first dose. In brief, rats were placed in a plastic box on a glass surface and allowed to habituate to the environment for 20 min. We used a radiant thermal stimulator (IITC Inc., Woodland Hills, CA), and the intensity of the light was adjusted at the start of the experiment so that average baseline latencies were approximately 9 s with a cut-off latency of 20 s. The focus of radiant thermal stimulation was aimed exactly on the middle of the plantar surface of each hind paw, and the latency to paw withdrawal was recorded. Three trials were performed at intervals of 5 min, and one score was assigned for each session by averaging the last two trials. The latencies were obtained alternately from the left and right hind paws, which were tested 5 min apart. The data were reported as the mean values for both the right and left hind paws combined. 2.5. Immunohistochemistry The rats were deeply anesthetized with pentobarbital (50 mg/ kg, i.p.) on day 7 or 14 after the start of paclitaxel (2 or 4 mg/kg) treatment, and the spinal cord was prepared for immunohistochemistry. Rats were perfused transcardially with 20 ml potassium-free phosphate-buffered saline (K þ -free PBS; pH 7.4) followed by 50 ml 4% paraformaldehyde solution. The spinal cord (L4–6) was removed, post-fixed for 3 h, cryoprotected overnight in 25% sucrose solution, and then stored at 80 °C until use. Spinal cords were cut at 15-μm thickness, thaw-mounted on silanecoated glass slides, and air-dried overnight at room temperature. Spinal cord sections were incubated with excess blocking buffer containing 2% skim milk in 0.1% Triton X-100 in K þ -free PBS and subsequently reacted overnight at 4 °C with the substance P Immunohistochemistry Staining Kit (Peninsula Laboratories LLC, San Carlos, CA) and rabbit polyclonal antiserum to CGRP (Enzo, Farmingdale, NY). The sections were then incubated in fluorescein isothiocyanate-conjugated anti-rabbit IgG (Sigma-Aldrich, Inc., St. Louis, MO, 1:200) for 2 h at room temperature. All sections were treated with Permafluor™ (Thermo Shandon, Pittsburgh, PA), coverslipped, and evaluated with microscopy. The immunostained sections were mounted on slides, covered with coverslips, and observed with an Olympus microscope (Olympus, Tokyo, Japan). The optical density (OD) of the stained sites was determined with ImageJ 1.46, an open source Java-based computer program. The OD of substance P and CGRP were calculated for days 7 and 14 after the start of paclitaxel or cremophor vehicle treatment. 2.6. Image analysis Signals were analyzed with fluorescence microscopy at 400 magnification using a microscopy digital camera system. Experimenters who were unaware of the experimental protocol observed the spinal cord sections. Five sections were randomly selected from each spinal cord (L4–6). The ratio of the area of substance P immunoreactivity to the area of CGRP immunoreactivity in the total profile was calculated for days 7 and 14 after the start of paclitaxel or vehicle treatment. Signal intensity and area frequency analysis of each neuron were calculated with ImageJ 1.46.
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2.7. Determination of substance P levels Immediately after sacrifice, the lumbar spinal cord was rapidly dissected out and frozen at 80 °C for substance P analysis. The lumbar spinal cord samples were cut into approximately 10-mm cubes and homogenized in a cold extraction buffer consisting of 20 mM Tris–HCl buffer at pH 8.0, with 100 mM Trizmas base, 400 mM NaCl, 4 mM EDTA, 1 mM PMSF, 7 mg/ml aprotinin, and 10% Triton X-100. The homogenates were vortexed for 2 min, centrifuged for 90 min at 42,000 g, and the supernatants were collected. The levels of substance P in the homogenates were measured with a competitive enzyme immunoassay (Substance P EIA kit; Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. The substance P EIA kit is a competitive assay that provides accurate measurement of substance P with a working range of 3.9–500 pg/ml, with a typical IC50 of approximately 30 pg/ml. 2.8. Statistical analysis All data are expressed as the mean 7S.E.M. The F-test followed by an unpaired t-test (Student's or Aspin-Welch's t-test) or oneway analysis of variance (ANOVA) followed by Dunnett's multiple comparison test were used to compare the effects of each paclitaxel dose and vehicle treatment. Statistical significance was established at P o0.05.
3. Results 3.1. The effects of paclitaxel on mechanical allodynia/hyperalgesia As expected, the mean paw withdrawal reflex was significantly increased with both 2 and 4 mg/kg paclitaxel treatment as determined with the von Frey test 7 and 14 days after the start of paclitaxel treatment compared to the cremophor vehicle (Fig. 1). On day 7, the responses to 2-g von Frey filament stimulation were significantly increased by 43.9 79.8% (2 mg/kg, P o0.01, n ¼ 6) and 51.77 12.4% (4 mg/kg, P o0.01, n ¼ 6) compared to vehicle treatment (9.57 3.6%, Fig. 1A). Paclitaxel also produced a significant increase on day 14, when the paw withdrawal incidences (%) for vehicle, 2, and 4 mg/kg paclitaxel treatment were 12.5 7 4.1%, 62.8 710.0% (P o0.01), and 66.2 712.7% (P o0.01), respectively. Similar results were obtained in response to 5-g von Frey filament stimulation. On day 7, withdrawal incidences for 2 mg/kg (58.7 77.6%) and 4 mg/kg treatment (68.37 7.2%) were both significantly increased compared to vehicle control (25.8 73.7%). On day 14, the responses to 5-g von Frey filament stimulation with paclitaxel (2 and 4 mg/kg) treatment were further increased compared to cremophor vehicle treatment. The response values were 91.1 77.8% (2 mg/kg) and 95.1 78.2% (4 mg/kg), and these were both significantly higher (P o0.01) than cremophor vehicle treatment (22.6 7 4.0%, Fig. 1B). 3.2. The effect of paclitaxel on thermal hyperalgesia We next investigated the effect of paclitaxel (2 and 4 mg/kg, i. p.) on thermal hyperalgesia. On day 7, the latency withdrawal time for thermal stimulation following cremophor vehicle treatment (7.83 70.50 s) was significantly decreased to 6.58 70.46 s (P o0.05, n ¼5) with paclitaxel (4 mg/kg) treatment. On day 14, the latency withdrawal time for thermal stimulation following paclitaxel (4 mg/kg: 5.747 0.33 s) treatment was further
Fig. 1. Effect of paclitaxel-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia as determined with the von Frey test and paw thermal test, respectively. (A, B) von Frey filaments (2 g and 5 g) were used to measure mechanical allodynia/hyperalgesia induced by paclitaxel treatment (2 and 4 mg/kg, i.p.; administered on days 0, 2, 4, and 6) in rats. Histograms show the mean 7 S.E.M. of the withdrawal response on days 7 and 14 after the start of paclitaxel or cremophor vehicle treatment. (C) The paw thermal test was used to measure thermal hyperalgesia induced by paclitaxel treatment (2 and 4 mg/kg, i.p.; administered on days 0, 2, 4, and 6) in rats. Histograms show the mean 7S.E.M. of the withdrawal response at days 7 and 14 after the start of paclitaxel and cremophor vehicle treatment. Data are the mean 7 S.E.M. of n ¼5–6 rats. *Po 0.05, **Po 0.01, one-way ANOVA with Dunnett’s post-hoc analysis compared to cremophor vehicle treatment.
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Fig. 2. Effects of paclitaxel treatment on substance P expression in the rat spinal cord. (A) Photomicrographs showing immunoreactivity for substance P in the lumbar dorsal horn of the spinal cord 14 days after the start of cremophor vehicle or paclitaxel (4 mg/kg) treatment. Scale bar, 100 μm. (B) OD of substance P immunostaining on days 7 and 14 after the start of cremophor vehicle or paclitaxel treatment. Histograms show the substance P OD in paclitaxel-treated (2 and 4 mg/kg) rats relative to cremophor vehicletreated rats. The blue box is the region used to obtain the OD. Data are the mean 7 S.E.M. n¼ 5 each for paclitaxel and cremophor vehicle treatment. One-way ANOVA with Dunnett’s post-hoc analysis compared to cremophor vehicle treatment. **Po 0.01 versus cremophor vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
decreased compared to cremophor vehicle treatment (7.55 70.30 s, P o0.01, n ¼ 5). Paclitaxel (2 mg/kg) treatment also significantly decreased the latency withdrawal time for thermal stimulation at 14 days (5.92 7 0.42 s, Po 0.01, n ¼5, Fig. 1C). 3.3. The effect of paclitaxel treatment on substance P expression and CGRP expression in superficial layers of the spinal dorsal horn Using immunohistochemistry, we detected substance P protein expression in the rat superficial layers of the spinal dorsal horn (layers I–II) at days 7 and 14 after the start of paclitaxel treatment. Compared with cremophor vehicle-treated rats, substance P protein expression was more intense after paclitaxel treatment (Fig. 2A). Using computerized OD image analysis, we measured the OD of superficial layers of the spinal dorsal horn that showed substance P protein expression. Paclitaxel (2 and 4 mg/kg) treatment induced a significant increase in the substance P OD in superficial layers of the spinal dorsal horn at day 14 (2 mg/kg: 24.07 2.4%; 4 mg/kg: 43.6 7 4.6%, P o0.01, n ¼5) compared with substance P protein expression in cremophor vehicle-treated rats (Fig. 2B). On the other hand, CGRP expression in the superficial layers of the spinal dorsal horn did not increase at day 14 following paclitaxel (4 mg/kg, n ¼5) treatment (Fig. 3A). Also, paclitaxel (4 mg/kg) treatment did not affect the CGRP OD in superficial layers of the spinal dorsal horn at days 7 and 14 (Fig. 3B). Thus,
substance P but not CGRP was significantly increased in the superficial layers of the spinal dorsal horn (layers I–II) in paclitaxeltreated rats. 3.4. The effect of paclitaxel treatment on substance P release in the spinal cord We removed the spinal cord at 14 days after the start of 2 and 4 mg/kg paclitaxel treatment. Release of substance P was quantified with a competitive enzyme immunoassay and compared with cremophor vehicle treatment. Substance P release was significantly increased both 2 mg/kg (700.0 7 67.8 pg/mg, P o0.05, n ¼5) and 4 mg/kg (959.3 725.4 pg/mg, P o0.01, n ¼ 4) paclitaxel treatment compared to cremophor vehicle treatment (524.3 780.9 pg/mg, Fig. 4). Thus, paclitaxel (2 and 4 mg/kg) treatment significantly increased the release of substance P in the spinal cord on day 14.
4. Discussion In the present study, mechanical allodynia/hyperalgesia and thermal hyperalgesia were observed on days 7 and 14 after the start of 2 or 4 mg/kg paclitaxel that was administered on 4 alternate days (from day 0 to day 6). When comparing mechanical
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Fig. 3. Effects of paclitaxel treatment on CGRP expression in the rat spinal cord. (A) Photomicrographs of CGRP in the lumbar dorsal horn of the spinal cord 14 days after the start of cremophor vehicle or paclitaxel (4 mg/kg) treatment. Scale bar, 100 μm. (B) OD of CGRP immunostaining on days 7 and 14 after the start of cremophor vehicle or paclitaxel treatment. Histograms show the CGRP OD in paclitaxel-treated (4 mg/kg) rats relative to cremophor vehicle-treated rats. The blue box is the region used to obtain the OD. Data are the mean 7 S.E.M. n¼ 5 each for paclitaxel and cremophor vehicle treatment. The F-test followed by the Student’s or Aspin-Welch’s t-test was used for evaluation of significance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Effect of paclitaxel treatment on the substance P level in the spinal cord. The histogram shows the level of substance P in the rat spinal cord 14 days after the start of paclitaxel (2 and 4 mg/kg, i.p.) treatment. Data are the mean 7 S.E.M. n ¼4– 5 each for cremophor vehicle treatment and paclitaxel treatment. One-way ANOVA with Dunnett’s post-hoc analysis compared to cremophor vehicle treatment. *Po 0.05, **P o0.01 versus cremophor vehicle.
allodynia/hyperalgesia and thermal hyperalgesia after administration of a single i.p., multiple i.p., or single i.v. dose of paclitaxel, no significantly different result was observed between the number of doses or the route at 7 days to 28 days after paclitaxel treatment (Itoh et al., 2004b; Polomano et al., 2001; Flatters and Bennett, 2004; Matsumoto et al., 2006). In our previous experimental studies, multiple doses of paclitaxel also produced mechanical allodynia/hyperalgesia and thermal hyperalgesia (Kawakami et al., 2012; Hara et al., 2013). Thus, the results of the present study are consistent with these previously published findings. Substance P and CGRP are excitatory neurotransmitters or neuromodulators that are released in the spinal cord dorsal horn by primary sensory afferents, thus contributing to the development of allodynia and hyperalgesia by facilitating the release of excitatory neurotransmitters from primary afferents (Ma and Eisenach, 2003). Moreover, Cata et al. (2006) have shown increased levels of excitability in spinal cord dorsal horn neurons in rats with paclitaxel-evoked neuropathic pain. Vachon et al. (2004) suggested a correlation between an increase in substance P and the development of mechanical allodynia induced by sciatic nerve cuff implantation. Our immunohistochemical analysis showed that paclitaxel-treated rats showed increased expression of substance P protein in the superficial layers of the spinal cord, in addition to development of mechanical allodynia/hyperalgesia. In contrast, CGRP was not changed in the spinal cord after paclitaxel treatment. Substance P is localized in high-threshold nociceptive c-fibers and is released by noxious stimuli (Otsuka and Yoshioka, 1993). Thus, paclitaxel-induced neuropathy is likely mediated by substance P, but not CGRP. We speculate that paclitaxel-induced neuropathic pain may be the result of up-regulation of substance P protein expression in the spinal cord. Also, paclitaxel increases the release of substance P from cultured adult rat DRG neurons
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(Tatsushima et al., 2011b). Regarding the mechanism, paclitaxel evokes the release of substance P from cultured DRG cells via an extracellular Ca2 þ influx through TRP channels (Tang et al., 2006; Tang et al., 2008; Miyano et al., 2009). In the present study, cross-reactivities of 97%, 93%, and 30% for the substance P fragments SP (4–11), SP (2–11), and SP (7–11), respectively, were observed with the substance P EIA kit. Moreover, cross-reactivities for substance P fragments SP (8–11) and SP (1–4) were less than 0.01%. Cross-reactivities were 12%, 2.7%, and 0.04% for Eledosin, neurokinin A, and neurokinin B, respectively. Several studies have demonstrated that substance P can be correctly analyzed using this substance P EIA kit (Itoh et al., 2004a, 2004b; Ursavas et al., 2007; Tatsushima et al., 2011b; Oz et al., 2011). Thus, we used this analysis method for studying paclitaxelinduced substance P release in the spinal cord and demonstrated that paclitaxel treatment significantly increased the release of substance P in the rat spinal cord. In addition, intrathecal injection of NK1 and NK2 receptor antagonists strongly reverses paclitaxelinduced neuropathy (Tatsushima et al., 2011b), and NK1 receptor antagonists relieve peripheral nerve injury-induced mechanical allodynia/hyperalgesia (Cahill and Coderre, 2002). We postulate that paclitaxel-induced substance P release in the DRG may facilitate substance P transport along central nerve terminals, resulting in an increase in substance P release in the dorsal horn of the spinal cord. These findings suggest that substance P released from the spinal cord is involved in paclitaxel-induced neuropathy. In conclusion, we suggest that paclitaxel-induced neuropathic pain may be the result of the release of substance P in the spinal cord dorsal horn.
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