Department of Applied Pharmacology, Graduate School of ...Sep 16, 2014 · JPET #217570 2 Running...
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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.)
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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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(min)
Ischemia Reperfusion
0 5 10 15 20
Blo
od
flo
w(m
L/m
in p
er
100
g)
0
10
20
30
40
50
60ControlIschemia-reperfusion
Figure 1
rubbertourniquet
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0 1 5 10
Hin
d-p
aw l
icki
ng
(sec
)
0
20
40
60
*
*
Ischemia time (min)
*
Cu
mu
lati
ve li
ckin
g(s
ec)
0
10
20
30
40
010 min 1020 min
**
2030 min
IR IR IR
Time after reperfusion (min)
0 5 10 15 20 25 30
Hin
d-p
aw li
ckin
g (
sec/
min
)
0
2
4
6
8IR () IR ()
A
B
C
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VH 1 3
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
50
**
Morphine (mg/kg)
VH 30 100
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
50
Gabapentin (mg/kg)
VH 10 30
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
50
Pregabalin (mg/kg)
A
B
C
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VH 100 200
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
50
*
NAC (mg/kg)
A
B
VH 30 100
Hin
d-p
aw l
icki
ng
(sec
)
0
10
20
30
40
50
**
HC-030031 (mg/kg)
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Time after injection (min)0 5 10
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
VH 0.1 0.3 Cu
mu
lati
ve li
ckin
g(s
ec)
0
50
100
150 *VH0.1% H2O2
*
H2O2 (%)
0.3% H2O2
A
VH 200
Hin
d-p
aw li
ckin
g(s
ec)
0
50
100
**
N-Acetyl-L-cystein(mg/kg)
VH 100
Hin
d-p
aw l
icki
ng
(sec
)
0
50
100
*
HC-030031(mg/kg)
B C
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PBN (g/site)
0 10 100
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
Saline PBN
Hin
d-p
aw li
ckin
g(s
ec)
0
10
20
30
40
50
100 g/site
***
**
A
B
H2O2-induced
IR-induced
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VH 30 100
Hin
d-p
aw l
icki
ng
(se
c)
0
10
20
30
*
*
BCTC (mg/kg)
Capsaicin-induced
VH 30 100
Hin
d-p
aw li
ckin
g(s
ec)
0
50
100
BCTC (mg/kg)
IR-induced
A
B
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VH CP VH CP
Nu
mb
er o
f w
ipin
g
0
20
40
60** *
Neo-VH Neo-CP
NS
CP-induced
Neo-VH Neo-CPHin
d-p
aw li
ckin
g(s
ec)
0
20
40
60IR-induced
A
B
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