Department of Applied Pharmacology, Graduate School of ...Sep 16, 2014 · JPET #217570 2 Running title: Peripheral Post-ischemic Dysesthesia in Mice Corresponding author: Yasushi

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A Mouse Model of Peripheral Post-Ischemic Dysesthesia: Involvement of

Reperfusion-Induced Oxidative Stress and TRPA1 Channel

Atsushi Sasaki, Shizuka Mizoguchi, Kenta Kagaya, Mai Shiro, Akiho Sakai, Tsugunobu

Andoh, Yurika Kino, Hiroyuki Taniguchi, Yukako Saito, Hiroki Takahata, and Yasushi

Kuraishi

Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical

Sciences, University of Toyama, Toyama, Japan (A.S., S.M., M.S., A.S., T.A., Y.K.);

Department I, Pharmacology Research Laboratories II, Research Division, Mitsubishi Tanabe

Pharma Corporation, Toda, Japan (K.K., Y.K., H.Tan.); Laboratory of Organic and

Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical

University, Sendai, Japan (Y.S., H.Tak.)

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 16, 2014 as DOI: 10.1124/jpet.114.217570

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Running title: Peripheral Post-ischemic Dysesthesia in Mice

Corresponding author: Yasushi Kuraishi

Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical

Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

TEL: +81-76-434-7510

Fax: +81-76-434-5045

E-mail: [email protected]

Text pages: 32

Tables: 0

Figures: 8

References: 49

Words

-Abstract: 215

-Introduction: 408

-Materials and Methods 1,029

-Results: 990

-Discussion: 1,382


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Abbreviations: ANOVA, analysis of variance; BCTC,

N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine- 1(2H)-carboxamide; I–R,

ischemia–reperfusion; i.p., intraperitoneal; ROS, reactive oxygen species; s.c., subcutaneous;

SEM, standard error of the mean; TRP, transient receptor potential

Recommended section: Inflammation, Immunopharmacology, and Asthma


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ABSTRACT

We behaviorally examined peripheral post-ischemic dysesthesia in mice and investigated the

underlying molecular mechanism with a focus on oxidative stress. Hind-paw ischemia was

induced by tight compression of the ankle with a rubber band, and reperfusion was achieved

by cutting the rubber tourniquet. We found that reperfusion after ischemia markedly provoked

licking of the reperfused hind paw, which was significantly inhibited by systemic

administration of the antioxidant N-acetyl-L-cysteine and the TRPA1 channel blocker

HC-030031. Post-ischemic licking was also significantly inhibited by an intraplantar injection

of another antioxidant phenyl-N-tert-butylnitrone. The TRPV1 channel blocker

N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide did

not inhibit post-ischemic licking. An intraplantar injection of hydrogen peroxide elicited

hind-paw licking, which was inhibited by N-acetyl-L-cysteine, phenyl-N-tert-butylnitrone, and

HC-030031. Post-ischemic licking was not affected by chemical depletion of sensory C-fibers,

but it was inhibited by morphine, which has been shown to inhibit the C- and Aδ-fiber-evoked

responses of dorsal horn neurons. Interestingly, post-ischemic licking was not inhibited by

gabapentin and pregabalin, which have been shown to inhibit the C-fiber- but not

Aδ-fiber-evoked response. The present results suggest that ischemia–reperfusion induces

oxidative stress, which activates TRPA1 channels to provoke post-ischemic licking. This

behavior is suggested to be mediated by myelinated (probably Aδ-type) afferent fibers.


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Oxidative stress and TRPA1 channels may be potential targets to treat peripheral

ischemia-associated dysesthesia.


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Introduction

Most people experience spontaneous thermal paresthesias (abnormal sensations),

spontaneous dysesthesias (unpleasant abnormal sensations), such as tingling, pricking, and

electric shock-like sensation, and in severe cases mechanical allodynia of the hand and foot

after sitting with their legs crossed or sleeping with an arm crooked under their head. Patients

with diabetic neuropathy or chemotherapy-induced neuropathy complain of spontaneous

dysesthesia (Bastyr et al., 2005; Driessen et al., 2012), and peripheral blood flow is decreased

in these neuropathies (Gauchan et al., 2009; Maxfield et al., 1997), raising the possibility that

ischemia is responsible for dysesthesia in these pathological conditions. Understanding the

mechanisms of post-ischemic dysesthesia may provide insight into the cellular and molecular

mechanisms underlying dysesthesia in neuropathic conditions, and this requires animal

models of peripheral post-ischemic dysesthesia. Because these sensations are felt superficially

in the skin (Merrington et al., 1949), we expected that post-ischemic dysesthesia would elicit

licking behaviors (post-ischemic licking) in animals. On the basis of this idea, we sought to

develop a mouse model of peripheral post-ischemic dysesthesia.

Myelinated Aβ-fibers are activated by light touches to the skin and discriminate texture

and object form, while myelinated Aδ-fibers and unmyelinated C-fibers respond to intense or

painful pressure (Delmas et al., 2011). Injury and diseases that affect the function of these

fiber subtypes lead to paresthesia and dysesthesia (Scherens et al., 2009). To characterize a


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mouse model of peripheral post-ischemic dysesthesia, we examined which types of primary

afferents would be involved in post-ischemic licking.

Peripheral post-ischemic dysesthesia occurs when blood supply returns (reperfusion) to a

previously ischemic tissue. Reperfusion following long-sustained ischemia causes severe

injury to cells in the target organs (Iida et al., 2009; Klune and Tsung, 2010). While the

pathogenesis of ischemia–reperfusion (I–R)-induced tissue injury is not completely

understood, I–R injury has been shown to be mediated by the generation of reactive oxygen

species (ROS), such as superoxide radical, hydroxyl radical and hydrogen peroxide (H2O2),

which results from the return of oxygen to the ischemic tissue (Zweier and Talukder, 2006).

Several lines of evidence suggest that ROS are involved in mechanical and thermal

hypersensitivities under inflammatory and neuropathic conditions (Fidanboylu et al., 2011;

Keeble et al., 2009). In addition, ROS generation has been shown to be involved in

itch-related response induced by intradermal injection of leukotriene B4 (Fernandes et al.,

2013). ROS may be associated with a variety of pathological cutaneous sensations. Thus, we

examined whether oxidative stress is responsible for peripheral post-ischemic licking.


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Materials and Methods

Animals. Male C57BL/6NCr mice (Japan SLC, Inc., Hamamatsu, Japan) were used. The

mice were 6 weeks old at the start of experiments and were housed in a room under controlled

temperature (21–23°C), humidity (45–65%), and lighting (lights on from 07:00 to 19:00 h)

conditions. Food and water were freely available. Experiments were conducted with the

approval of the Animal Care Committee of the University of Toyama and according to the

guidelines for investigations of experimental pain in animals published by the International

Association for the Study of Pain (Zimmermann, 1983).

Agents. N-acetyl-L-cysteine (Sigma-Aldrich, St. Louis, MO) and phenyl-N-tert-

butylnitrone (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were dissolved in

physiological saline. The transient receptor potential (TRP) A1 channel blocker

2-(1,3-Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamid

e (HC-030031; Sigma-Aldrich) was dissolved in physiological saline containing 10%

dimethyl sulfoxide and 5% Tween-80.

N-(4-tert-Butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)- carboxamide

(BCTC; Enzo Life Sciences, Plymouth Meeting, PA) was dissolved in regular tap water

containing 0.5% carboxymethyl cellulose and 1% Tween-80. Morphine hydrochloride

(Sankyo, Tokyo, Japan) was dissolved in physiological saline; the weight of morphine refers


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to the salt. Gabapentin (synthesized by H.T. and Y. S.) and pregabalin (Sequoia Research

Products Limited, Pangbourne, UK) were dissolved in regular tap water. Capsaicin

(Sigma-Aldrich) was dissolved in physiological saline containing 7.5% dimethyl sulfoxide

(for local injection and ocular instillation) or in physiological saline containing 10% ethanol

and 10% Tween 80 (for systemic administration). A solution of 30% H2O2 (Wako Pure

Chemical Industries, Osaka, Japan) was diluted to 0.1% or 0.3% with physiological saline.

Morphine has been shown to have antiallodynic effects at subcutaneous (s.c.) doses of 1

to 5 mg/kg in mice, peaking at 15–30 min after injection (Takasaki et al., 2000). Therefore,

morphine (1 and 3 mg/kg) was administered s.c. 15 min before the start of compression

ischemia. Gabapentin (100 mg/kg, s.c.) and pregabalin (30 mg/kg, s.c.) have been shown to

have antiallodynic effects in mice, peaking 1–2 h after injection (Field et al., 2006). Therefore,

gabapentin (30 and 100 mg/kg) and pregabalin (10 and 30 mg/kg) were administered s.c. 60

min before the start of compression ischemia. HC-030031 has been shown to inhibit

hyperalgesia at intraperitoneal (i.p.) doses of 30–300 mg/kg, peaking at 1–2 h after injection

(da Costa et al., 2010). Therefore, HC-030031 (30 and 100 mg/kg) was administered i.p. 60

min before the start of compression ischemia or intraplantar H2O2 injection.

Capsaicin-induced hyperalgesia has been shown to be inhibited by 30-min pretreament with

BCTC (30 mg/kg, oral) and Freund's complete adjuvant-induced hyperalgesia has been shown

to be inhibited by 2-h pretreatment with BCTC (30 mg/kg, oral) in rats (Pomonis et al., 2003).


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Therefore, BCTC (30 and 100 mg/kg) was administered orally 60 min before the start of

compression ischemia or intraplantar capsaicin injection. The doses of N-acetyl-L-cysteine

and phenyl-N-tert- butylnitrone were chosen from our preliminary experiments in which the

effects of these agents were examined on licking induced by intraplantar injection of H2O2

solution.

Compression Ischemia of the Hind Paw. To induce hind-paw ischemia, a glabrous

region just proximal to the ankle joint was firmly compressed with a cut rubber band (Oband,

#16, Kyowa Limited, Osaka, Japan) for 10 min (Fig. 1) except for a series of experiments in

which the ankle was compressed for 1 or 5 min. Except for blood-flow assessment (see

below), ischemic compression was performed in unanesthetized mice. Reperfusion was

achieved by cutting the rubber tourniquet.

Assessment of Hind-Paw Blood Flow. Mice were anesthetized with pentobarbital (70

mg/kg, i.p.) and their body temperature was maintained at 36.5–37.5°C using a

thermostatically-controlled heating pad coupled to a rectal probe (BWT-100; Bio Research

Center Co., Ltd., Nagoya, Japan). A laser Doppler flowmeter (ALF21; Advance Co., Ltd.,

Tokyo, Japan) was used to assess blood flow in the plantar skin of the hind paw. The probe

(Type N, 0.5 mm diameter) of the laser Doppler flowmeter was held 1 mm from the plantar


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surface to avoid mechanical and thermal effects on blood flow. Flux signals were low pass

filtered with a time constant of 3 seconds to reduce movement artifacts.

Post-Ischemic Licking. Mice were placed individually in the observation chambers (8 ×

10 × 20 cm) with an acrylic transparent floor. After a 30-min acclimation period, the mice

underwent compression ischemia for 10 min as described above. Immediately after cutting the

rubber tourniquet, the mice were placed back into their chambers and their behaviors were

videotaped with no one present. The time spent licking the treated hind paw (see Fig. 1) was

measured with a stopwatch during video playback. The mice shook the treated hindlimb

following compression-reperfusion, which subsided after a few minutes; this behavior was not

quantified in this study.

Agent-Induced Licking. Mice were individually placed in the observation chamber for

at least 30 min to adapt to the environment. Solutions of capsaicin (0.1 µg) and H2O2 were

injected into the plantar region of the unilateral hind paw at a volume of 20 µL. The mice

were immediately put back into their chamber and their behaviors were videotaped with no

one present. The time spent licking the treated paw was measured with a stopwatch during

video playback (Sasaki et al., 2013).


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Neonatal Capsaicin Treatment. Capsaicin (50 mg/kg) or vehicle was injected s.c. twice

on day 2 and 5 after birth (Nakano et al., 2008). To verify sensory C-fiber neuron depletion,

one drop (10 µL) of 0.1% capsaicin or vehicle was applied to both eyes, and the number of

eye wiping with the forelimbs within 30 seconds was counted (Nakano et al., 2008).

Data Analysis. Data are presented as mean ± standard error of the mean (SEM). For

licking behavior, statistical differences between two groups were analyzed with Student's

t-test or Mann-Whitney rank sum test. Statistical differences among three or more groups

were analyzed with one-way analysis of variance (ANOVA) or Kruskal–Wallis one-way

ANOVA on ranks (for data without normal distribution or equal variance) followed by post

hoc Dunnett's test. For wiping behavior, statistical differences were analyzed with two-way

ANOVA followed by post hoc Tukey's test. A P value of less than 0.05 was considered

significant. Statistical analyses were performed using Sigmaplot graphing and statistical

software (version 11.2; Systat Software, Inc., San Jose, CA).


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RESULTS

Changes in Plantar Skin Blood Flow during Ankle Compression and after

Reperfusion. Tight compression of the ankle with a rubber tourniquet produced prompt (< 1

min) and almost complete occlusion of blood flow to the hind paw (Fig. 1). The loss of blood

flow persisted during the 10-min compression (Fig. 1). After cutting the tourniquet, blood

flow was promptly increased up to > 200% of the pre-compression value within 1 min (Fig. 1).

This reactive hyperemia gradually decreased and almost subsided at 10 min after tourniquet

cutting. There were no significant changes in hind-paw blood flow during the 25-min period

in control mice that did not undergo ankle compression (Fig. 1).

Post-Ischemic Licking. When untreated control mice were returned to the observation

chamber, they demonstrated active exploratory behavior and increased locomotor activity for

about 10 min, with rare licking of the hind paws (Fig. 2A). In contrast, mice demonstrated

marked licking of the treated hind paw during the first 10-min period following I–R (Fig. 2A).

The cumulative time of hind-paw licking was significantly (Mann-Whitney rank sum test; P =

0.004) increased during the first 10-min period as compared with that in control animals (Fig.

2B). During the 10–30-min period after I–R, licking of the hind paws was observed mainly as

a series of grooming behaviors, and the time spent hind-paw licking was similar between the

I–R and control groups (Fig. 2A and B). These results suggest that hind-paw licking during


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the first 10-min period was primarily due to I–R. Then, we investigated the effects of duration

of ischemia (1, 5, and 10 min) on the I–R-induced hind-paw licking during the first 10-min

period. The cumulative time of hind-paw licking was significantly (one-way AOVA; F3,22 =

9.753, P <0.001) increased after compression ischemia in an ischemic duration-dependent

manner (Fig. 2C). Since the time of hind-paw licking was longest after 10-min ischemia, in

the subsequent experiments, we investigated the pharmacological characteristics and

mechanisms of post-ischemic licking during the first 10-min period after 10-min I–R.

Effects of Morphine, Gabapentin, and Pregabalin on Post-Ischemic Licking.

Hind-paw licking following I–R was dose-dependently and significantly (Kruskal–Wallis

one-way ANOVA on ranks; P = 0.001) inhibited by s.c. injections of morphine (1 and 3

mg/kg) (Fig. 3A). The licking was almost completely inhibited at a dose of 3 mg/kg (Fig. 3A).

In contrast, neither gabapentin (30 and 100 mg/kg, s.c.) nor pregabalin (10 and 30 mg/kg, s.c.)

inhibited post-ischemic licking (Fig. 3B and C).

Effects of N-Acetyl-L-Cysteine and HC-030031 on Post-Ischemic Licking. The

antioxidant N-acetyl-L-cysteine has been used as a tool for investigating the role of ROS in

numerous biological and pathological processes (Zafarullah et al., 2003). To determine

whether reperfusion-induced oxidative stress is involved in post-ischemic licking, we


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investigated the effect of N-acetyl-L-cysteine on post-ischemic licking. N-Acetyl-L-cysteine

(100 and 200 mg/kg, i.p.) dose-dependently and significantly (Kruskal–Wallis one-way

ANOVA on ranks; P = 0.002) inhibited post-ischemic licking, which was almost completely

inhibited at 200 mg/kg (Fig. 4A).

HC-030031 is a potent and selective TRPA1 channel blocker and has been used as a tool

for investigating the role of TRPA1 channel in both in vitro and in vivo experiments

(Fernandes et al., 2013; McNamara et al., 2007). To determine whether TRPA1 channels are

involved in post-ischemic licking, we investigated the effect of HC-030031. HC-030031 (30

and 100 mg/kg, i.p.) dose-dependently and significantly (one-way ANOVA; F2,21 = 6.234, P =

0.007) inhibited post-ischemic licking (Fig. 4B).

H2O2-Induced Hind-Paw Licking and its Suppression by N-Acetyl-L-Cysteine and

HC-030031. Intraplantar injections of 0.1% and 0.3% H2O2 concentration-dependently and

significantly (Kruskal–Wallis one-way ANOVA on ranks; P <0.001) elicited hind-paw licking

(Fig. 5A). We observed that licking peaked within 1 min after 0.1% H2O2 injection and then

rapidly subsided. Licking after 0.3% H2O2 injection persisted for a longer period and

decreased more slowly. Licking induced by 0.3% H2O2 was significantly (Student's t-test; P =

0.009) inhibited by pretreatment with N-acetyl-L-cysteine (200 mg/kg, i.p.) (Fig. 5B).

H2O2-induced licking was also significantly (Student's t-test; P = 0.014) inhibited by


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pretreatment with HC-030031 (100 mg/kg, i.p.) (Fig. 5C).

Effects of Local Injection of Phenyl-N-tert-Butylnitrone on Licking Induced by H2O2

Injection and Ischemia-Reperfusion. We examined the effects of intraplantar injection of

another antioxidant phenyl-N-tert-butylnitrone (Das et al., 2012) on licking induced by H2O2

injection and ischemia-reperfusion in order to confirm the role of ROS production in the paw

distal to the compression site. Intraplantar injections of phenyl-N-tert-butylnitrone (10 and

100 µg/site) together with 0.3% H2O2 dose-dependently and significantly (one-way ANOVA;

F2,18 = 11.661, P <0.001) inhibited H2O2-induced licking, with a significant inhibition

(Dunnett's test; P <0.05) observed at a dose of 100 µg/site (Fig. 6A). An intraplantar injection

of phenyl-N-tert-butylnitrone (100 µg/site) significantly (Student's t-test; P =0.006) inhibited

post-ischemic licking also (Fig. 6B).

Effects of TRPV1 Channel Blocker on Capsaicin-Induced and Post-Ischemic

Licking. An intraplantar injection of capsaicin (0.1 μg) elicited hind-paw licking; the effect

peaked within 1 min and then subsided in a few minutes (data not shown). Thus, the

cumulative time spent licking was measured for 3 min after injection. The selective TRPV1

channel blocker BCTC (Pomonis et al., 2003) dose-dependently and significantly (one-way

ANOVA; F2,15 = 8.548, P = 0.003) inhibited capsaicin-induced licking at oral doses of 30 and


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100 mg/kg (Fig. 7A). In contrast, there was an increased tendency for post-ischemic licking

after BCTC administration at the same doses but this apparent difference was not statistically

significant (one-way ANOVA; F2,15 = 1.585, P = 0.237) (Fig. 7B).

Effect of Neonatal Capsaicin Treatment on Post-Ischemic Licking. Mice underwent

neonatal capsaicin treatment to deplete sensory C-fiber neurons expressing TRPV1 channels.

A wiping test was performed to confirm the effectiveness of the treatment. Neonatal capsaicin

treatment significantly reduced capsaicin-induced eye wiping (Fig. 8A); two-way ANOVA

revealed significant main effect of neonatal capsaicin treatment (F1,12 = 9.437, P = 0.01) and

significant interaction between neonatal capsaicin treatment and eye stimuli (F1,12 = 5.11, P =

0.043). However, neonatal capsaicin treatment did not significantly affect post-ischemic

licking (Fig. 8B).


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DISCUSSION

In human experiments, peripheral post-ischemic dysesthesias begin approximately 1 min

after release of cuff compression of 5–30 min duration and persist for 5–10 min (Merrington

and Nathan, 1949; Suarez-Roca et al., 2003). Following I–R of the leg, we generally

experience spontaneous dysesthesias such as tingling and pricking, and in severe cases tactile

stimulation of the toes can elicit pain. In the present study, we found that reperfusion

following a 10-min arrest of blood flow to the hind paw induced licking of the ipsilateral hind

paw for approximately 10 min. Because mice could freely move on their extremities, the

hind-paw licking might be due to spontaneous and touch-evoked dysesthesias; thus, the

behavior is a potential indication of acute post-ischemic dysesthesias in mice.

After release of compression ischemia, blood flow was promptly increased up to >200%

of the pre-compression value within 1 min and gradually decreased for about 10 min.

Re-establishment of blood flow in the ischemic tissue restores the supplies of oxygen and

glucose that are indispensable for cell survival. However, mitochondria and oxidative

stress-promoting enzymes, such as nicotinamide adenine dinucleotide phosphate oxidases also

utilize oxygen, and activation of these generator systems leads to increased ROS generation

(Zweier and Talukder, 2006). ROS oxidize proteins, lipids, and intracellular ions (Moran et al.,

2001). In the present study, intraplantar injection of H2O2 elicited hind-paw licking, and this

behavior was significantly inhibited by i.p. administration of the antioxidant


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N-acetyl-L-cysteine. Similarly, post-ischemic licking was inhibited by N-acetyl-L-cysteine. In

addition, an intraplantar injection of the antioxidant phenyl-N-tert-butylnitrone inhibited

post-ischemic licking at a dose that suppressed H2O2-induced licking. These results, taken

together, suggest that oxidative stress induced by I–R is involved in post-ischemic licking and

that the paw distal to the compression site is a causal tissue.

In the present study, the selective TRPA1 channel blocker HC-030031 inhibited

post-ischemic licking. The selective TRPV1 channel blocker BCTC did not inhibit

post-ischemic licking at a dose that significantly suppressed licking induced by capsaicin, a

selective TRPV1 channel agonist. These results suggest that activation of TRPA1, but not

TRPV1, plays a key role in post-ischemic licking. Several lines of evidence indicate that

TRPA1 channels are a major oxidation sensor. Various oxidants, such as tert-butyl

hydroperoxide, potassium superoxide, H2O2, and 4-hydroxynonenal, directly activate TRPA1

channels (Andersson et al., 2008; Bessac et al., 2008; Sawada et al., 2008). Oxidation of

intracellular cysteine residues is critical for TRPA1 channel activation. Six cysteine residues

located in the amino-terminal segment have been identified as potential sites for oxidation and

activation of TRPA1 channel (Takahashi and Mori, 2011). Cysteine-reducing agents almost

completely inhibit TRPA1-mediated Ca2+ influx elicited by H2O2 (Sawada et al., 2008). In

contrast, cysteine-oxidizing agents elicit TRPA1-mediated Ca2+ influx (Sawada et al., 2008).

H2O2 is known to oxidize cysteine residues in proteins to form either cysteine sulfenic acids or


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disulfides (Poole et al., 2004). In the present study, H2O2-induced paw licking was inhibited

by HC-030031 as well as N-acetyl-L-cysteine, indicating that H2O2 elicits licking via the

activation of TRPA1 channel. Our findings suggest that reperfusion-induced oxidative stress

causes post-ischemic licking via TRPA1 channel activation. On the other hand, TRPV1

channels are activated by capsaicin, noxious heat, and low pH (Caterina et al., 2000) but not

oxidants, such as H2O2 and 4-hydroxynonenal (Andersson et al., 2008; Bessac et al., 2008).

TRPA1 channels are expressed in myelinated A-fiber, as well as unmyelinated C-fiber sensory

neurons (Kobayashi et al., 2005; Kwan et al., 2009). Direct administration of H2O2 to dorsal

root ganglion neurons elicits TRPA1-mediated Ca2+ influx (Andersson et al., 2008; Sawada et

al., 2008). Taken together, these findings suggest that ROS directly activates TRPA1 channels

in primary sensory neurons to elicit post-ischemic licking. Since TRPA1 channels are also

present in keratinocytes in the skin (Kwan et al., 2009), their activation in keratinocytes may

elicit and/or modulate the excitation of primary sensory neurons that are involved in

post-ischemic dysesthesias.

TRPA1 channels have been also proposed to be involved in mechanotransduction. In

TRPA1-deficient mice, the mechanically evoked firing rate is reduced in both slowly adapting

C-fiber nociceptors and Aδ-fiber mechanoreceptors (Kwan et al., 2009). TRPA1 deficiency

also reduces the firing rate of slowly adapting Aβ-fibers (Kwan et al., 2009). In contrast to the

reduced firing in all subclasses of slowly adapting fibers, TRPA1 deficiency increases


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mechanically evoked firing in rapidly adapting hair follicle afferents, including D-hair and

rapidly adapting Aβ-fibers (Kwan et al., 2009). These findings raise the possibility that

subclasses of slowly adapting fibers are involved in the perception of tactile dysesthesia. Thus,

we examined the roles of myelinated A-fibers and unmyelinated C-fibers in mediating

post-ischemic licking by degenerating the latter via neonatal capsaicin injections. This

treatment is known to cause permanent degeneration of most C-fibers but does not affect

A-fibers (Nakano et al., 2008; Sasaki et al., 2008). We found that post-ischemic licking was

unaffected by neonatal capsaicin treatment, suggesting that this behavior is mediated by

A-fibers.

Morphine, a µ-opioid receptor agonist, inhibited post-ischemic licking. µ-Opioid

receptors are expressed in C- and Aδ-fiber, but not Aβ-fiber, sensory neurons (Sasaki et al.,

2008; Yamamoto et al., 2008). It has been reported that morphine inhibits the activity of rat

dorsal horn neurons evoked by stimulation of C- and Aδ-fibers but not that evoked by

stimulation of Aβ-fibers (Jurna et al., 1979). Considering that ablation of C-fibers by neonatal

capsaicin treatment did not affect post-ischemic licking, Aδ-fibers are likely responsible for

post-ischemic licking behavior. In the present study, gabapentin (30 and 100 mg/kg) and

pregabalin (10 and 30 mg/kg) did not inhibit post-ischemic licking. It has been reported that

s.c. injections of gabapentin (100 mg/kg) and pregabalin (30 mg/kg) markedly inhibit

formalin-induced nociceptive behaviors and nerve ligation-induced mechanical allodynia in


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mice (Field et al., 2006). Gabapentin and pregabalin inhibit C-fiber-evoked responses of rat

dorsal horn neurons without affecting Aδ-fiber-evoked responses (Tanabe et al., 2006; You et

al., 2009). Thus, the present results that gabapentin and pregabalin did not inhibit

post-ischemic licking support the hypothesis that Aδ-fibers are responsible for post-ischemic

licking.

Pricking and tingling are recognized as separate sensations (Ochoa and Torebjörk, 1980;

Suarez-Roca et al., 2003). Both are localized to the skin, but pricking is very intense, almost

painful, whereas tingling is a less intense sensation with marked rhythmicity (Ochoa and

Torebjörk, 1980; Suarez-Roca et al., 2003). Electrophysiological recordings of human sensory

fiber activity following the release of compression ischemia of the arm have suggested that

pricking and tingling sensations arise in thinly myelinated Aδ and large myelinated Aβ

afferent fibers, respectively (Ochoa and Torebjörk, 1980). Thus, pricking sensations may be

more responsible for post-ischemic licking than tingling sensations.

Oxidative stress occurs in neuropathic conditions, including diabetic peripheral

neuropathy and chemotherapy-induced peripheral neuropathy (Head, 2006), which are

accompanied by mechanical allodynia and spontaneous dysesthesias such as pricking and

tingling sensations (Bastyr et al., 2005; Driessen et al., 2012). It has been shown that ROS

increases in diabetes and that oxidative stress is involved in diabetic peripheral neuropathy

(Singh et al., 2014). ROS production by the anticancer drug paclitaxel has been demonstrated


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in cultures of HeLa cells and cortical neuronal cells (Jang et al., 2008; Kim et al., 2008).

Paclitaxel also increases the number of atypical (swollen and vacuolated) mitochondria in

sensory axons, suggesting mitochondrial injury in neuropathic conditions (Flatters and

Bennett, 2006; Jin et al., 2008). The pivotal role of ROS in neuronal injuries has been further

demonstrated by the beneficial impact of N-acetyl-L-cysteine therapy in the treatment of

diabetic neuropathy in rats (Kamboj et al., 2010) and neuropathy induced by oxaliplatin, a

chemotherapeutic agent, in humans (Lin et al., 2006). Peripheral blood flow is decreased in

conditions of diabetic neuropathy (Maxfield et al., 1997) and neuropathy induced by

paclitaxel and oxaliplatin (Gauchan et al., 2009). Taken together, these findings suggest that

sudden increase in peripheral blood flow in these pathological conditions causes dysesthesia

and allodynia through activation of the ROS-TRPA1 pathway.

In summary, our results demonstrate that reperfusion after brief hind-paw ischemia

induces hind-paw licking following by transient reactive hyperemia in mice. Moreover, our

findings suggest that reperfusion-induced oxidative stress activates TRPA1 channels to

provoke peripheral post-ischemic licking, which is mediated by myelinated (probably

Aδ-type) afferent fibers. Targeting oxidative stress with antioxidants or inhibiting TRPA1

channels using selective TRPA1 channel blockers may be novel and effective therapeutic

options for peripheral ischemia-associated allodynia and dysesthesias.


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Authorship contributions

Participated in research design: Sasaki, Andoh, Taniguchi, and Kuraishi

Conducted experiments: Sasaki, Mizoguchi, Kagaya, Shiro, Sakai, Kino

Contributed new reagents and analytic tools: Saito and Takahata

Performed data analysis: Sasaki

Wrote or contributed to the writing of the manuscript: Sasaki, Andoh, and Kuraishi


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FOOTNOTES

This work was supported in part by the Grant-in-Aid from the Nakatomi Foundation (to

A.S.).

Hiroki Takahata is a deceased co-author (died in March, 2014).


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FIGURE LEGENDS

Fig. 1. Changes in plantar skin blood flow during ankle compression and after reperfusion. In

four mice, the ankle was firmly compressed with a rubber band for 10 min, and then the

rubber tourniquet was cut for reperfusion. Four control mice did not undergo ankle

compression. Plantar skin blood flow was measured with a laser Doppler flow meter. Data are

presented as mean ± SEM. The upper diagram illustrates a rubber tourniquet and a region

(gray in color), licking of which was measured as post-ischemic responses.

Fig. 2. Ischemia–reperfusion (I–R) induces hind-paw licking. After ankle compression, the

rubber tourniquet was cut and mice were immediately placed in the observation chamber. The

time spent licking the hind paw following I–R was measured with a stopwatch. A,

Time-course of hind-paw licking after 10-min ischemia and subsequent reperfusion. Data are

presented as mean ± SEM (n = 6 each). B, The cumulative time of hind-paw licking at 10-min

intervals during 30 min after 10-min ischemia and subsequent reperfusion. Data are presented

as mean ± SEM (n = 6 each). **P < 0.01 (Mann-Whitney rank sum test). C, Effects of ankle

compression duration on hind-paw licking. The ankle was compressed for 1, 5, or 10 min, and

the time spent licking the hind paw was measured for 10 min. Data are presented as mean ±


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SEM (n = 7, 6, 6, and 7 for ischemia time of 0, 1, 5, and 10 min, respectively). *P < 0.05 vs.

time 0 (Dunnett's test).

Fig. 3. Effect of morphine, gabapentin, and pregabalin on post-ischemic licking. Morphine (1

and 3 mg/kg), gabapentin (30 and 100 mg/kg), and pregabalin (10 and 30 mg/kg) were

administered subcutaneously 15, 60, and 60 min before the start of compression ischemia,

respectively. The time spent licking the treated hind paw was measured for 10 min following

10-min ischemia and subsequent reperfusion. Data are presented as means ± SEM (n = 6

each). *P < 0.05 vs. vehicle (VH) (Dunnett's test).

Fig. 4. Effects of systemic injections of antioxidant and TRPA1 channel blocker on

post-ischemic licking. The antioxidant N-acetyl-L-cysteine (NAC; 100 and 200 mg/kg) or the

TRPA1 channel blocker HC-030031 (30 and 100 mg/kg) was administered intraperitoneally

20 or 60 min before the start of compression ischemia, respectively. The time spent licking the

treated hind paw was measured for 10 min following 10-min ischemia and subsequent

reperfusion. Data are presented as means ± SEM (n = 6 and 8 for A and B, respectively). *P <

0.05 vs. vehicle (VH) (Dunnett's test).


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Fig. 5. Effects of systemic injections of antioxidant and TRPA1 channel blocker on

H2O2-induced licking. A, H2O2-induced licking. H2O2 (0.1 and 0.3%) or vehicle (VH) was

injected intraplantarly, and the time spent licking the treated paw was measured for 10 min

after injection. *P< 0.05 vs. VH (Dunnett's test). B, Effect of the antioxidant

N-acetyl-L-cysteine on H2O2-induced licking. N-Acetyl-L-cysteine (200 mg/kg) and VH were

administered intraperitoneally 20 min before 0.3% H2O2 injection. **P < 0.01 (Student's t-test).

C, Effect of the TRPA1 channel blocker HC-030031 on H2O2-induced licking. HC-030031

(100 mg/kg) and VH were administered intraperitoneally 60 min before 0.3% H2O2 injection.

*P < 0.05 (Student's t-test). Data are presented as means ± SEM (n = 8 for A and n = 6 for B

and C).

Fig. 6. Effects of local injection of antioxidant on licking induced by H2O2 injection and

ischemia–reperfusion (I–R). The antioxidant phenyl-N-tert-butylnitrone (PBN) was injected

intraplantarly together with 0.3% H2O2 (A) or alone immediately after the start of

compression ischemia (B). The time spent licking the treated hind paw was measured for 10

min following H2O2 injection (A) or 10-min ischemia and subsequent reperfusion (B). The

broken line represents the average value of the saline-injected group (n = 7). Data are

presented as means ± SEM (n = 7 each). *P < 0.05 vs. control without PBN injection

(Dunnett's test). **P < 0.01 (Student's t-test).


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Fig. 7. Effects of TRPV1channel blocker on capsaicin-induced and post-ischemic licking. A,

Capsaicin-induced licking. Capsaicin (0.1 µg) was injected intraplantarly, and the time spent

licking the treated paw was measured for 3 min after injection. B, Post-ischemic licking. The

mice underwent 10-min compression ischemia and reperfusion, and the time spent licking the

treated paw was measured for 10 min after ischemia–reperfusion (I–R). The TRPV1 channel

blocker N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-

carboxamide (BCTC) was administered orally 60 min before capsaicin injection or the start of

compression ischemia. Data are presented as means ± SEM (n = 6 each). *P < 0.05 vs. VH

(Dunnett's test).

Fig. 8. Effect of neonatal capsaicin treatment on post-ischemic licking. Neonatal mice were

treated with capsaicin (Neo-CP) or vehicle (Neo-VH). A, Capsaicin instillation-induced

forelimb wiping. One drop (10 µl) of 0.1% capsaicin (CP) or vehicle (VH) was instilled into

both eyes, and the number of forelimb wiping was counted for 30 seconds after instillation.

**P < 0.01, ***P < 0.001 (Tukey's test). B, Post-ischemic licking. The time spent licking the

treated hind paw was measured for 10 min after 10-min ischemia and subsequent reperfusion

(I–R). Data are presented as means ± SEM (n = 4 each).


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JPET Fast Forward. Published on September 16, 2014 as DOI: 10.1124/jpet.114.217570

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Documents

Department of Applied Pharmacology, Graduate School of ...Sep 16, 2014 · JPET #217570 2 Running title: Peripheral Post-ischemic Dysesthesia in Mice Corresponding author: Yasushi