Inhibition of Paclitaxel-induced A-fiber...

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Inhibition of Paclitaxel-induced A-fiber-Hypersensitization

by Gabapentin

Misaki Matsumoto, Makoto Inoue, Andreas Hald, Weijiao Xie

and Hiroshi Ueda

Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate

School of Biomedical Sciences, Nagasaki 852-8521, Japan (M.M., M.I., A.H., W.X.,

H.U.)

JPET Fast Forward. Published on May 10, 2006 as DOI:10.1124/jpet.106.103614

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 10, 2006 as DOI: 10.1124/jpet.106.103614

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a) Running title: Gabapentin inhibits paclitaxel-induced neuropathic pain.

b) Address for correspondence: Dr. Hiroshi Ueda,


School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

Tel: +81-95-819-2421; Fax: +81-95-819-2420; Email: [email protected]

c) Number of text pages, figures and references:

Text pages: 27

Figures: 4

Tables: 0

References: 40

Number of words in

-Abstract: 224

-Introduction: 344

-Discussion: 752

d) Abbreviations: EPW, electrical-stimulus induced paw withdrawal; DRG, dorsal root

ganglion; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; Caα2δ-1, voltage-gated

calcium channel α2δ-1subunit.

e) Recommended section: Neuropharmacology


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Abstract

Paclitaxel (Taxol®) is a widely used chemotherapeutic agent in the treatment of several

tumors. However, its use is often associated with the generation of peripheral neuropathic

pain expressed as mechanical allodynia and thermal hyperalgesia. The molecular

mechanism behind this debilitating side effect is obscure and efficient drugs for its

prevention are required. We sought to clarify the cellular changes in the involved

nociceptor types underlying paclitaxel-induced neuropathic pain, and to test for an

alleviating effect of gabapentin treatment in a murine model of paclitaxel-induced

neuropathic pain. We found that a single treatment with paclitaxel (4 mg/kg, i.p.) led to a

decrease in both the thermal and mechanical nociceptive thresholds, as well as reducing

the thresholds for 250 Hz (Aδ-fiber) and 2000 Hz (Aβ-fiber) but not 5 Hz (C-fiber)

sine-wave electrical stimuli-induced paw withdrawal (EPW). The paclitaxel-induced

neuropathic pain was completely abrogated by gabapentin (30 mg/kg, i.p.) treatment.

Furthermore, we found that mRNA and protein levels of the calcium channel subunit

α2δ-1 (Caα2δ-1), one of the putative targets for gabapentin, was upregulated in dorsal

root ganglions (DRG), as well as increased expression of Caα2δ-1 protein in

medium/large DRG neurons by immohistochemistry, following paclitaxel treatment.

This suggests, that paclitaxel induces A-fiber specific hypersensitization, which may

contribute to the functional mechanical allodynia and hyperalgesia, and that gabapentin

could be a potential therapeutic agent for paclitaxel-induced neuropathic pain.


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Introduction

Paclitaxel (Taxol®) is widely used in cancer chemotherapy for the treatment of solid

carcinomas (Kohler and Goldspiel, 1994; Socinski, 1999). Its mechanism is known to

involve an increase in the stability of tubulin polymers and inhibit cellular replication

(Schiff et al., 1979; Horwitz, 1992). However, it has serious side effects such as

peripheral neuropathic pain (Rowinsky et al., 1993; Chaudhry et al., 1994; Cavaletti et al.,

1995; Forsyth et al., 1997), which become more marked with continued therapy. In some

cases, the side effects may outweigh the beneficial effects on life quality, necessitating a

dose reduction (Bristol-Myers Squibb Company, 2003). However, the molecular

mechanism underlying paclitaxel-induced neuropathic pain is enigmatic and no

therapeutics for its prevention are known.

Recently, the anticonvulsant gabapentin has proven effective in the treatment of

neuropathic pain, and in neuropathic pain models such as the peripheral nerve ligation

and diabetic neuropathic pain model, gabapentin inhibits thermal hyperalgesia and tactile

allodynia (Hunter et al., 1997; Hwang and Yaksh, 1997; Cesena and Calcutt, 1999; Miki

et al., 2001; Luo et al., 2002). Although gabapentin was originally designed as a structural

analogue of the neurotransmitter GABA, it was revealed that gabapentin binds with

high-affinity to Caα2δ-1 (Gee et al., 1996), which most likely accounts for its analgesic

effects (Cheng and Chiou, 2006). Furthermore, gabapentin has been reported to inhibit

high-threshold Ca2+ channels and modulate synaptic neurotransmission (Patel et al.,

2000; Shimoyama et al., 2000; Sutton et al., 2002; Maneuf et al., 2004). In addition, Luo

et al. (2002) reported that gabapentin efficacy is correlated with the increase of Caα2δ-1

in DRG in peripheral nerve injury models.

In the present study, we demonstrated that gabapentin inhibits


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paclitaxel-induced peripheral neuropathic pain in mice. Furthermore, Neurometer tests,

which are known to determine nociceptor sensitivity, as well as to distinguish between the

different types of sensory neurons in humans and rodents (Masson and Boulton, 1991;

Pitei et al., 1994; Kiso et al., 2001), showed an A-fiber specific hypersensitization

following paclitaxel treatment. Finally, we report an increased expression of Caα2δ-1 in

medium/large DRG neurons after paclitaxel treatment.


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Methods

Animals

Male ddY mice weighing 20 - 22 g were used. They were kept in a room maintained at 21

± 2ºC with free access to standard laboratory diet and tap water. Procedures were

approved by Nagasaki University Animal Care Committee and complied with the

recommendations of the International Association for the Study of Pain (Zimmermann,

1983).

Drugs

The following drugs were used: Paclitaxel (Taxol®) was generously provided by

Bristol–Myers Squibb Company, Paris, France. Paclitaxel was dissolved in saline just

before administration. Gabapentin [1-(Aminomethyl)-cyclohexaneacetic acid] was

purchased from Sigma Chemical (St Louis, MO, U.S.A.), dissolved in saline.

Drug injection and analysis

We used three types of paclitaxel (4 mg/kg) -injection schedule as follows; single i.p.

injection, multiple (four times on alternate days; days 0, 2, 4 and 6) i.p. injection and

single i.v. injection. In the “area under the curve” (AUC) analysis for paclitaxel effect on

pain thresholds, we calculated the area under the curve generated by the paw withdrawal

threshold (PWT)-time course from week 1 to 4 after paclitaxel treatment using a

trapezoidal method. Gabapentin (3 – 30 mg/kg) was injected (i.p.) 30 minutes before

behavioral tests.

Conventional nociceptive tests

In thermal paw withdrawal tests, nociception was measured as the latency to paw

withdrawal evoked by exposure to a thermal stimulus (Hargreaves et al., 1988; Inoue et


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al., 2004). Unanesthetized animals were placed in plexiglas cages on top of a glass sheet

and an adaptation period of 1 hour was allowed. The thermal stimulator (IITC Inc.,

Woodland Hills, CA, USA) was positioned under the glass sheet and the focus of the

projection bulb was aimed exactly on the middle of the plantar surface of the animal. A

mirror attached to the stimulator, permitted visualization of the plantar surface. A cut-off

time of 20 seconds was set in order to prevent tissue damage. The paw pressure test was

performed as described previously (Rashid et al., 2003; Inoue et al., 2004). Briefly, mice

were placed into a plexiglas chamber on a 6×6 mm wire mesh grid floor and were

allowed to acclimatize for a period of 1 hour. The mechanical stimulus was then delivered

onto the middle of the plantar surface of the right hind-paw using a Transducer Indicator

(Model 1601, IITC Inc., Woodland Hills, USA). The pressure needed to induce a flexor

response was defined as the pain threshold. All behavioral experiments were carried out

by investigators blinded to the drug-treatment.

Electrical stimulation-induced paw withdrawal (EPW) test

Electrodes (Neurotron Inc., Blatimore, MD) were fastened to the right plantar surface and

instep of the mice. Transcutaneous nerve stimuli with each of the three sine-wave pulses

(5, 250 and 2000Hz) were applied using the Neurometer CPT/C (Neurotron Inc.). The

minimum intensity (µA) at which each mouse withdrew its paw was defined as the

current stimulus threshold. Stimuli were applied with 10 minutes intervals. All behavioral

experiments were carried out by investigators blinded to the drug-treatment.

Quantitative real-time PCR

On the 3rd, 7th and 14th day after paclitaxel treatment, the L4-6 DRGs were removed and

RNA was purified using TRIzol (Invitrogen, CA, USA). Total RNA (1 µg) was used for

cDNA synthesis with Superscript II reverse transcriptase and random hexamer primers


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(Invitrogen, CA, USA). Real-time quantitative PCR (RT-PCR) was performed using an

ABI Prism 7000 Sequence Detection system (Applied Biosystems, Tokyo, Japan), using

qPCR MasterMix Plus for SYBR® Green I (Eurogentec, Seraing, Belgium) containing

dNTPs (+dUTP), Hot Goldstar DNA polymerase, and Urasil-N Glycosilase, according to

the manufactures instructions. The cycling conditions for all primers were the following:

10 minutes at 95°C to activate the Hot Goldstar DNA polymerase, followed by 50 cycles

consisting of two steps, 15 seconds at 95°C (denaturation) and 1 minute at 60°C

(annealing-extension). The Caα2δ-1mRNA levels were evaluated by comparison to

glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences were as

follows; Caα2δ-1, (F)5′-CTC ATG CAA GCG GAA CAG ACT TCT-3′ and (R)5′-TCA

AAG CAG ACA TCA GGT CCT TT-3′ corresponding to the mouse Caα2δ-1 gene (NCBI

accession number; NM_009784) (Martin et al., 2002), GAPDH, (F)5′- TAT GAC TCC

ACT CAC GGC AAA T-3′ and (R)5′-GGG TCT CGC TCC TGG AAG AT-3’

corresponding to the mouse GAPDH (NCBI accession number; NM_001001303). In all

cases, the validity of amplification was confirmed by the presence of a single peak in the

melting temperature analysis and linear amplification against the PCR cycles.

Western blot analysis

On the 7th, 14th and 28th day after paclitaxel treatment, the L4-6 DRGs were removed

for Western blot analysis. Mixed L4-6 DRG protein (30 µg) were applied to an

SDS-polyacrylamide gel (8 %). The anti- Caα2δ-1 antibody (Sigma, USA) diluted 1:200

(Inoue et al., 2004) was used and anti-β−Tubulin antibody (Santa Cruz Biotechnology,

CA, USA) diluted 1:1000 was used as internal control. HRP-labeled rabbit antibody was

used as 2nd antibody at 1:2000. Visualization of immunoreactive bands was performed


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by the Light Capture (AE-6960/C/FC, Atto, Tokyo, Japan) with an enhanced

chemiluminescent substrate for the detection of horseradish peroxidase, SuperSignal®

West Pico Chemiluminescent Substrate (PIERCE, Rockford, IL). The intensities of

immunoreactive bands were analyzed by NIH imaging for Macintosh.

Immunohistochemistry

The immunohistochemical analysis were performed as described previously (Inoue et al.,

2004). Briefly, mice were deeply anesthetized with sodium pentobarbital (50 mg/kg i.p.)

and perfused transcardially with 20 ml of potassium-free phosphate-buffered saline

(K+-free PBS, pH 7.4) followed by 50 ml of 4% paraformaldehyde solution. The L4-6

DRGs were removed, postfixed for 3 hours, and cryoprotected overnight in 25% sucrose

solution. The DRGs were fast-frozen in cryo-embedding compound on a mixture of

ethanol and dry ice and stored at -80°C until use. DRGs were cut at 10 µm thickness,

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

temperature (RT). Before immunolabeling, antigen unmasking was performed by

microwave treatment for 15 minutes in10 mM citrate buffer (pH 6.0). DRG sections were

incubated with excess blocking buffer containing 2% skim milk in PBST (0.1% Triton

X-100 in K+-free PBS) and subsequently reacted overnight at 4°C with anti-

Caα2δ-1antibodies (1:200; Sigma, USA) in 2% BSA/PBST. The sections were then

placed in fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:200; Santa

Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 hours at RT. All sections were treated

with Permafluor (Thermo Shandon, Pittsburgh, PA) and coverslipped and evaluated by

microscopy (Keyence, Tokyo, Japan).

Measurements of DRG neuron diameters were done using the BZ Image


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Measurement software (Keyence, Tokyo, Japan). Total cell counts was carried out in

bright field images as reported elsewhere (Josephson et al., 2001; Dai et al., 2004).

Therefore, the quantification of the data may represent a biased estimate of the actual

number of neurons. However, in order to determine the Caα2δ-1 positive neuron profile,

we have carried out the counts using only neurons with clearly visible nuclei. In this

experiment, we divided neurons into 2 groups, small and medium/large neurons. We used

24 µm in diameter as dividing line between small and medium/large neurons (Gold MS et

al., 1996; Hiruma et al., 2000). The total 1684 DRG neurons from vehicle-treated mice

(n=3) and 2724 DRG neurons from paclitaxel-treated mice (n=4) were measured.

Statistical analysis

The behavioral time course data were analyzed using a Student’s t-test to detect

differences between vehicle- and paclitaxel- treated groups. The behavioral data on

gabapentin and the RT-PCR data as well as the western blot data of Caα2δ-1 were

analyzed using a one-way ANOVA and Tukey multiple comparison post-hoc analysis.

The criterion of significance was set at p<0.05. All results were expressed as mean ±

S.E.M.


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Results

Paclitaxel-induced thermal hyperalgesia and mechanical allodynia.

We treated mice with paclitaxel (4 mg/kg) single (i.p. or i.v.) or multiple times (four i.p.

injections on alternate days; days 0, 2, 4 and 6), and evaluated the threshold in several

nociceptive tests from pre-injection (control) to 4 weeks after the first treatment. No

significant side effects such as ascites, loss of body weight or alopecia was observed in

this experimental schedule. A single i.p. injection of paclitaxel (4 mg/kg) led to a

decreased threshold in the paw pressure test, which was significant at 1 and 2 weeks after

treatment but increased to baseline level at week 4 post treatment (Fig. 1A). When

comparing the mechanical allodynia after a single i.p. injection, multiple i.p. injections or

single i.v. injection of paclitaxel, no significantly different outcomes were observed,

between the numbers of administrations or the route, as shown as the AUC values for the

paw pressure test from week 1 to 4 after treatment (Fig. 1B). The thermal nociceptive

threshold level decreased in a manner similar to the decrease in threshold for mechanical

allodynia, though the effect was less pronounced. However, the effect was significant 2

weeks after a single i.p. injection of paclitaxel (Fig. 1C). Based on these results, we

determined to adopt the single i.p. injection for the paclitaxel-induced neuropathic pain

model in mice and to evaluate the effect of gabapentin 2 weeks after treatment which was

the peak time point of neuropathic pain.

Blockade of paclitaxel-induced neuropathic pain by gabapentin.

Gabapentin, injected i.p. 30 minutes before behavioral tests, inhibited mechanical

allodynia 2 weeks after paclitaxel-treatment (4 mg/kg, single i.p. injection) in a

dose-dependent manner from 3 to 30 mg/kg, while gabapentin (30 mg/kg) did not cause


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any significant effects in vehicle-treated mice (Fig. 1D). The duration of the analgesic

effect from treatment with 30 mg/kg gabapentin lasted for more than 24 hours but the

significant effect had vanished by 48 hours after treatment (Fig. 1E). Moreover,

gabapentin injection (30 mg/kg, i.p.) 2 weeks after paclitaxel-treatment reversed thermal

hyperalgesia within 30 minutes. However, gabapentin treatment of vehicle-treated mice

did not affect the thresholds of thermal paw withdrawal latency (Fig. 1F).

Paclitaxel-induced myelinated A-fiber specific hypersensitization

Transcutaneous nerve stimuli with each of the three sine-wave pulses of different

frequencies of 5, 250 and 2000 Hz to activate C-, Aδ- and Aβ-fibers, respectively

(Katims, 1997; Lengyel et al., 1998), were applied to the right hind paw of the mice, and

the intensity (µA) was gradually increased. Thresholds were determined, at which paw

withdrawal was observed, from pre-injection (control) to 4 weeks after paclitaxel

treatment (Fig. 2A). The reduction in threshold by the 250 Hz (Aδ) and 2000 Hz (Aβ)

stimuli at 1 and 2 weeks after paclitaxel-treatment (4 mg/kg, single i.p. injection), had

completely vanished 4 weeks after treatment. No significant change in the threshold by 5

Hz (C) stimuli was observed at any time. These results suggest that paclitaxel induces

myelinated Aδ and Aβ-fiber specific hypersensitization.

Blockade of paclitaxel-induced myelinated A-fiber specific hypersensitization by

gabapentin.

Gabapentin injection (30 mg/kg, i.p.) 2 weeks after paclitaxel-treatment reversed the

hyperalgesia within 30 minutes as determined by 250 Hz (Aδ) and 2000 Hz (Aβ)

stimulations. However, gabapentin treatment of vehicle-treated mice did not affect the


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thresholds of neither 250 Hz (Aδ) and 2000 Hz (Aβ) stimuli induced responses (Fig. 2B).

Increased expression of Caα2δ-1 mRNA and protein in DRG after paclitaxel

treatment.

We removed L4-6 DRGs at the 3rd, 7th and 14th day after paclitaxel treatment and

Caα2δ-1 mRNA expression was quantified by RT-PCR analysis and compared to

GAPDH mRNA expression. The specificity of the PCR primers was confirmed by the

occurrence of a single peak in the melting curves for the PCR products, and the efficiency

of the reaction was found to be approximately 100 % shown by a threshold cycle (Ct) vs.

sample-dilution plot (Fig. 3A). Caα2δ-1 mRNA expression was slightly but not

significantly increased at day 3 but a larger and significant increase was observed at day 7

in paclitaxel-treated mice compared to controls (Fig. 3A). At day 14, the Caα2δ-1 mRNA

expression was reduced to control level.

Moreover, we removed L4-6 DRGs at 7th, 14th and 28th day after paclitaxel

treatment and Caα2δ-1 protein expression was quantified by western blot analysis. The

result indicated that Caα2δ-1 up-regulation was significant at day 7, maximum at day 14

and returned to baseline level 28 days post paclitaxel injection (Fig. 3B).

Increased expression of Caα2δ-1 in medium/large sized DRG neurons following

paclitaxel treatment.

We detected Caα2δ-1 protein expression in DRGs at day 14 after treatment using

immunohistochemistry. In vehicle-treated mice, Caα2δ-1 expression was mainly

localized to small diameter DRG neurons (Fig. 4A), while in paclitaxel-treated mice,


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Caα2δ-1 protein expression was both observed in small diameter DRG neurons and in

medium/large diameter DRG neurons (Fig. 4A). The total amount of neurons in the

DRGs, which stained positive for Caα2δ-1 protein, was counted and their individual sizes

measured both in paclitaxel- and vehicle- treated mice. A significant increase in the

frequency of stained neurons was found in the paclitaxel-treated mice compared to

vehicle-treated controls (Fig. 4B). Furthermore the cell-size measurements showed that

the increased number of Caα2δ-1 stained neurons reflected an increased expression of

Caα2δ-1 in medium/large sized neurons, while the number of positively stained small

neurons remained almost same between vehicle- and paclitaxel- treated groups (Fig. 4C

and D). However, no significant difference in the general size distribution of DRG

neurons was found between the two groups, supporting the notion, that the increased

Caα2δ-1 protein expression indeed resulted from an up-regulation in medium/large sized

neurons (Fig. 4C).


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Discussion

Here we found that single administration of paclitaxel at a dose of 4 mg/kg i.p. caused

peripheral neuropathic pain. According to the paclitaxel injection prescribing information

sheet, the recommended dose is 135-175 mg/m2 (Bristol-Myers Squibb Company, 2003),

which is converted into 3.6-4.7 mg/kg approximately (Height 1.7 m, Weight 65 kg; Du

Bois formula). In addition, it states that paclitaxel should be given every 3 weeks in

clinical states. However, in previous experimental studies, the paclitaxel administration

has been performed multiple times on consecutive/alternate days or in a single but high

dose (Authier et al.,2000; Dina et al., 2001; Polomano et al., 2001; Flatters and Bennett,

2004; Smith et al., 2004). Therefore, the present finding seems to have some advantages.

We conclude that the protocol using single administration of paclitaxel (4 mg/kg) is

proper and easy approach to study paclitaxel-induced neuropathic pain.

In our experimental model, we observed mechanical allodynia, thermal

hyperalgesia and A-fiber specific hypersensitization using the Neuromater test. The

Neurometer test is widely used clinically in the evaluation of sensory function in humans

with complex regional pain syndrome or other types of peripheral neuropathic pain,

which can result from common diseases such as diabetes mellitus (Masson and Boulton,

1991; Katims, 1997; Lengyel et al., 1998). Our results are supported by previous data

from animal models of paclitaxel-induced hypersensitivity (Smith et al., 2004), as well as

neurometer analysis of paclitaxel-treated patients, which also show Aβ-fiber

hypersensitivity, as determined by stimulating the median nerve with 2000 Hz pulses

(Doi et al., 2003). Therefore, we conclude that our model is useful for investigating the

mechanism by which paclitaxel induces neuropathic pain and to test new drug candidates.


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Frequent reports of the alleviating effect of gabapentin treatment in various

models of neuropathic pain such as the peripheral nerve ligation model and diabetic

neuropathic pain models, are published (Hunter et al., 1997; Hwang and Yaksh, 1997;

Cesena and Calcutt, 1999; Miki et al., 2001; Luo et al., 2002). The present study is the

first to report an anti-nociceptive effect of gabapentin on paclitaxel-induced neuropathic

pain in mice. We evaluated the effect by EPW tests as well as conventional nociceptive

tests measuring mechanical nociception. Gabapentin revealed specific analgesic effects

against paclitaxel-induced mechanical allodynia, thermal hyperalgesia and A-fiber

hypersensitization without affecting the pain threshold of naïve mice. These results

suggest that gabapentin could be used to treat paclitaxel-induced neuropathic pain in the

clinic.

Referring to the mechanism of anti-nociceptive effects of gabapentin, we

measured the expression of Caα2δ-1, which is known to possess gabapentin-binding

properties (Gee et al., 1996). We first demonstrated that the expression of Caα2δ-1

mRNA and protein was increased in the DRGs of paclitaxel-treated mice. The difference

in the peak time point between mRNA and protein expression seems to be attributable to

the longer life-time of protein. It should be noted that the time course of Caα2δ-1 protein

expression was very close to that of paclitaxel-induced neuropathic pain (Fig. 1A, 1C and

Fig. 2A). Interestingly, immunohistochemistry revealed that Caα2δ-1 protein was

increased in medium/large diameter neurons following paclitaxel-treatment. As the

function of the calcium channel containing Caα2δ-1 is related to modulation of synaptic

neurotransmission (Patel et al., 2000; Shimoyama et al., 2000; Maneuf et al., 2004),

up-regulation of Caα2δ-1 expression in medium/large-sized neurons may contribute to


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the paclitaxel-induced mechanical allodynia and A-fiber dependent hypersensitization.

Recently, we have reported that Caα2δ-1 is upregulated in medium/large-sized DRG

neurons following peripheral nerve injury or intrathecal injection of lysophosphatidic

acid, an initiator of neuropathic pain (Inoue et al., 2004). Luo et al. (2002) reported that

inhibitory effects of gabapentin on neuropathic pain correlates to Caα2δ-1 expression.

Gabapentin sensitivity and Caα2δ-1 up-regulation were observed in spinal nerve ligation,

spinal nerve transection, sciatic nerve chronic constriction injury and diabetic models, but

not in the vincristine-induced neuropathic pain model. In the present study, we confirmed

the gabapentin sensitivity and Caα2δ-1 up-regulation in the paclitaxel-induced

neuropathic pain model. It is interesting that the gabapentin sensitivity and Caα2δ-1

up-regulation differed between the vincristine and paclitaxel models, when taken into

account that both drugs are known to act through tubulin assembly. Although the

underlying mechanism remains to be determined, it might be attributed different manners

of action; disruption of tubulin assembly by vincristine and increasing stabilization by

paclitaxel (Kohler and Goldspiel, 1994).

In conclusion, we first report, that paclitaxel induces A-fiber hypersensitization

and novel expression of Caα2δ-1 in medium/large DRG neurons, which may functionally

contribute to mechanical allodynia and hyperalgesia. Furthermore, we identify

gabapentin as a potential therapeutic agent for paclitaxel-induced peripheral neuropathic

pain.

Acknowledgements


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We thank N. Uminotaira and A. Yamaguchi for technical help and behavioral

studies. Paclitaxel (Taxol®) was generously provided by Bristol–Myers Squibb Company,

Paris, France.


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Footnotes

a) Financial assistance: Part of this study was supported by the Grant-in-Aid for the

Cancer Research (15-7) from the Ministry of Helth, Labour, and Welfare.

b) Reprint requests should be addressed to: Dr. Hiroshi Ueda,


School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

Tel: +81-95-819-2421; Fax: +81-95-819-2420; Email: [email protected]


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Legends for Figures

Fig. 1. Paclitaxel-induced neuropathic pain and antinociceptive effect of gabapentin.

(A) Time course of paclitaxel-induced mechanical allodynia from pre-injection (control)

to 4 weeks post-treatment. (B) Summation of paclitaxel-induced mechanical allodynia

determined as the AUC from week 1 to 4 in response to a single i.p. injection, single i.v.

injection or 4 i.p. injections on alternate days (multi). (C) Time course of

paclitaxel-induced thermal hyperalgesia from pre-injection to 4 weeks after treatment.

(D) Dose-dependent analgesic effects of gabapentin in mechanical paw pressure tests 2

weeks after paclitaxel treatment. (E) Time course of gabapentin (30 mg/kg)-induced

analgesia 2 weeks after paclitaxel treatment (4 mg/kg, i.p.). (F) Analgesic effects of

gabapentin in thermal paw withdrawal tests 2 weeks after paclitaxel treatment (4 mg/kg,

i.p.). Animals were analyzed 30 minutes after gabapentin (30 mg/kg) treatment: control

(C), saline (Sal) and gabapentin (GBP), *:p<0.05, **:p<0.01 vs. control (vehicle).

#:p<0.05, ##:p<0.01 vs. paclitaxel/saline. Data are presented as mean ± S.E.M. from

experiments using at least 6 mice.

Fig. 2. Paclitaxel-induced A-fiber hypersensitization and inhibitory effects of gabapentin.

(A) Time courses of paclitaxel (4 mg/kg, i.p.)-induced sensory fiber sensitizations

determined by EPW tests using Neurometer from pre-injection to 4 weeks after treatment.

(B) Alleviating effects of gabapentin on paclitaxel-induced A-fiber hypersensitivity

determined by EPW tests using 250 Hz and 2000 Hz stimulations 2 weeks after paclitaxel

treatment. All animals were analyzed 30 minutes after gabapentin treatment: Control (C),

saline (Sal) and gabapentin (GBP). *:p<0.05, **:p<0.01 vs. control or vehicle/saline.

##:p<0.01 vs. paclitaxel/saline. Data are presented as mean ± S.E.M. from experiments


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using at least 6 mice.

Fig. 3. Expression of Caα2δ-1 mRNA and protein in L4-6 DRGs after

paclitaxel-treatment. (A. Left and Middle) Dissociation curves of PCR products from

reactions using primers specific for mRNA encoding Caα2δ-1 and GAPDH, and

amplification efficiency analysis. (A. Right) Quantitative RT-PCR analysis. Caα2δ-1 and

GAPDH mRNAs in L4-6 DRGs on 3rd, 7th and 14th day after paclitaxel treatment were

measured and Caα2δ-1 was normalized to GAPDH. The graph shows the relative amount

of Caα2δ-1 mRNA in paclitaxel-treated mice compared to the controls. (B) Western blot

analysis. Caα2δ-1 and β-Tubulin protein in L4-6 DRGs 7, 14 and 28 days after paclitaxel

treatment were measured and Caα2δ-1 was normalized to β-Tubulin. The graph shows

the relative amount of Caα2δ-1 protein in paclitaxel-treated mice compared to the

controls. Photograph shows representative data. *:p<0.05, **:p<0.01 vs. control (C).

Data are presented as mean ± S.E.M. from experiments using at least 8 mice.

Fig. 4. Increased expression of Caα2δ-1 protein in medium/large DRG neurons. (A) DRG

slices of 10 µm from day 14 after vehicle or paclitaxel treatment were stained with

antibodies against Caα2δ-1. (B) The percentage of Caα2δ-1 stained neurons in paclitaxel-

and vehicle-treated mice 2 weeks after treatment. The total 1684 DRG neurons from

vehicle-treated mice (n=3) and 2724 DRG neurons from paclitaxel treated mice (n=4)

were measured. (C) Size distribution of all neurons (total) and of Caα2δ-1 expressing

neurons in DRG in paclitaxel- and vehicle-treated mice, respectively. (D) Bar graph

represents the frequency distribution of Caα2δ-1 positive neurons normalized to cell size.


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Line graph (solid line) represents the balance value of the vehicle- and paclitaxel- treated

group. Scale bar, 50 µm. *:p<0.05 vs. vehicle.


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