Category: Research Paper
Spinal p38 Activity and Analgesic Effect after Low and Highintensity Electroacupuncture Stimulation in a Plantar Incision Rat Model
Sheng-Feng Hsua,b,1, Yen-Jing Zenga,c,1, Shih-Ying Tsaid, Kuen-Bao Chene, Julia Yi-Ru Chenf,g, Ju-Hsin Changc,e, Yeong-Ray Wend,e,h
a Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical
University, Taichung, Taiwan
b Department of Acupuncture, China Medical University Hospital Taipei Branch, Taipei,
Taiwan
c Graduate Institute of Clinical Medical Science, College of Medicine, China Medical
University, Taichung, Taiwan
d Department of Anesthesiology, School of Medicine, China Medical University, Taichung,
Taiwan
e Department of Anesthesiology, China Medical University Hospital, Taichung, Taiwan f Department of Pediatrics, School of Medicine, Taipei Medical University, Taipei, Taiwan g Guang Li Biomedicine, Inc. ,Xizhi, New Taipei City, Taiwan
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h Research Center for Chinese Medicine and Acupuncture, China Medical University, Taichung, Taiwan
1 S.-F. Hsu and Y.-J. Zeng have equal contributions as the first authors in this study.
Corresponding Author:
Yeong-Ray Wen, M.D., Ph.D.
Department of Anesthesiology, China Medical University Hospital
No. 2, Yuh-Der Rd, North District, 40447, Taichung, Taiwan
Tel.: +886-4-22052121 ext 3562;
Fax: +886-4-22052121 ext 3598.
*E-mail: yr.wen@yahoo.com.tw; yrwen@mail.cmu.edu.tw 20
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Abstract
Aims: Postoperative pain is a major problem. Electroacupuncture (EA) has been accepted as a
useful and low-risk complementary therapy for post-operative pain. Animal studies indicate that surgical incision activates p38 MAPK in the spinal microglia, which critically contributes to post-incisional nociceptive development. How EA affects incision-induced p38 activation is important but yet to be fully elucidated.
Methods: Male adult rats received plantar incision (PI) at the right hind paw followed by 30-
min EA of 4-Hz, one of two intensities (3 and 10 mA), and at right ST36 (Zusanli) acupoint immediately after PI and for 3 successive days. EA analgesia was evaluated by von Frey fibers and Hargreaves’ tests. Spinal p38 activation was examined by immunostaining. In separate groups, SB203580, a p38 inhibitor, was intrathecally injected alone or with EA to test the combining effect on nociception and spinal phospho-p38.
Key findings: EA of 10-mA significantly ameliorated mechanical allodynia, but 3-mA did
not. None of them altered thermal hyperalgesia. Repeated EA could not inhibit phospho-p38 in the PI rats, contrarily, EA per se significantly induced phospho-p38 in the normal rats.
Intrathecal SB203580 injection dose-dependently prevented PI-induced allodynia.
Combination of low-dose SB203580 and 3-mA EA, which were ineffective individually, profoundly reduce post-PI allodynia.
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Significance: We demonstrated that 10-mA EA exerts a significant inhibition against post-PI
mechanical hypersensitivity via a p38-independent pathway. Importantly, co-treatment with low-dose p38 inhibitor and 3-mA EA can counteract spinal phospho-p38 to exert strong analgesic effect. Our finding suggests a novel strategy to improve EA analgesic quality.
Keywords: electroacupuncture (EA); p38 MAPK; postoperative pain; Zusanli (ST36); spinal cord
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Introduction
Post-operative pain is a common acute pain problem. Poorly controlled pain increases the risks of heart attack, pneumonia, deep vein thrombosis, immune impairment, anxiety, and persistent neuropathic pain (Kehlet et al. 2006; Liu and Wu 2007). Standard opioid administration remains the mainstay (Momeni et al. 2006), however, its use is complicated with opioid-related side effects which degrade analgesic quality. Our and other human studies demonstrated perioperative acupuncture or electroacupuncture (EA) could simultaneously reduce postoperative opioid consumption, spare adverse symptoms, and meanwhile maintain adequate analgesia (J. G. Lin et al. 2002; S. M. Wang et al. 2008b). Although EA has been accepted to pose these advantages, its use is still unpopular due to unclear mechanisms and low therapeutic efficacies.
Opioid-dependent and opioid-independent mechanisms interactively underlie acupuncture-induced analgesia (Han 2003; Sun et al. 2008; Shu Ming Wang et al. 2008a;
Wen et al. 2010). Following acupuncture stimulation, neuron-based releases of neuropeptides, including endogenous opiates-like endorphin, enkephalin, dynorphin, and endomorphin (Cabyoglu et al. 2006; Fukazawa et al. 2005; Han 2004, 2003; Han and Terenius 1982; Yun Wang et al. 2005); non-opioid substances like serotonin (Baek et al.
2005; Cabyoglu et al. 2006; Chang et al. 2004; J. G. Lin and Chen 2008), noradrenaline (Cabyoglu et al. 2006; Chang et al. 2004; Wen et al. 2010), oxytocin (Yang et al. 2007), 58
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neuropeptide Y (Lee et al. 2009), neurotrophin-3 (Mi et al. 2011), somatostatin (Dong et al.
2005), spinal orexin A (Feng et al. 2012), and newly found peripheral release of adenosine (Goldman et al. 2010) were all involved to play roles in regulating acupuncture effects.
However, how these molecules participate and interact in various pain processes are complex and unclarified.
On the other hand, different lines of studies proved that neuroglia are powerful contributors or modulators in persistent pain states (Gosselin et al. 2010; Ji et al. 2009). Using a plantar incision (PI) model, which imitates postoperative pain as a preclinical animal study (Brennan et al. 1996), we showed that activation of p38 mitogen-activated protein kinase (MAPK) within microglia in the spinal dorsal horn contributed to nociceptive development at early post-PI stage (Wen et al. 2009). Pretreatment with p38 inhibitor suppressed phosphorylated-p38 (p-p38), an activated form of p38, can dose-dependently prevented behavioral hypersensitivity and downstream proinflammatory cytokines and chemokines (Lu et al. 2013; Wen et al. 2009). Emerging evidence indicated that EA effect may be partly mediated through microglial regulation by inhibiting intracellular signaling, including p-p38, and exocytosis of inflammatory mediators in many pain conditions (Gim et al. 2011; L. L.
Liang et al. 2010; Y. Liang et al. 2012; Mi et al. 2011; Shan et al. 2007; Sun et al. 2006; K.
D. Xu et al. 2010). Therefore, it would be of great value to investigate how EA controls p38 activation in surgical pain.
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The present animal study aimed to answer the following questions: first, if EA could ameliorate PI-induced nociceptive behaviors; second, if EA suppresses incision-induced p- p38 in spinal microglia; and third, if combination of EA and p38 inhibitor could enhance analgesic effect.
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Materials and Methods Experimental Animals
Adult male Sprague–Dawley rats (200–250 g; BioLASCO, Taiwan) were tri-housed and allowed to freely access food and water. The animal facility maintained a 12-h light-dark cycle at 22–24C and humidity of 70%. Animal managements were approved by Institutional Animal Care and Use Committee of China Medical University and followed the institutional Animal Care Guidelines. Efforts were made to minimize animal number and suffering.
Anesthesia, PI model, and EA
Experiments design and protocols were referring to our previous PI and EA studies (Brennan et al. 1996; Wen et al. 2009; Wen et al. 2007). In brief, the rats were anesthetized with 2%
isoflurane in oxygen via a nasal mask. An one-cm longitudinal incision was made at plantar surface of the right hind paw, 0.5 cm from the edge of the heel, through skin, plantar fascia, until flexor digitorum brevis muscle. The skin wound was sutured by skin layers with 50 nylon. Then the rats were transferred to transparent holders and were continuously anesthetized with 1% isoflurane in 100% oxygen via a cone nasal mask for EA stimulation.
EA was conducted at Zusanli (ST36) of right hind limb by a pair of 36G (0.22 mm in diameter) acupuncture needles with electric stimulation generated from a Grass S88 electrostimulator (Astromed, Grass, West Warwick, RI, USA) via constant current units 101
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(Grass CCU1A, West Warwick, RI, USA). Stimulation parameters were set at 4-Hz square waves of 0.5 msec pulse width for a period of 30 minutes, following our lab protocol (Wen et al. 2007). Two intensities, 3 mA (low-intensity) and 10 mA (high-intensity), were applied to rats according to the group allocation. ST36 is the most frequently used acupoint and has been shown to produce systemic analgesic effects in animals (Pomeranz et al. 1977; Wen et al. 2007). The rats in the sham EA groups received needle insertion in muscle layer (5 mm depth) of ST36 but without electrostimulation. All rats were removed from the anesthetic apparatus after stop of EA, and all of them rapidly recovered to a movable state within 12 min. The recovery was so fast that anesthesia would not interfere the first behavioral test conducted 1 h later. We had shown this manipulation did not change behaviors or induce Fos expression (Wen et al. 2010).
Behavioral tests
Animals were acclimatized to experimental environment from at least 2 days prior to study.
To test mechanical threshold, the rats were put in inverted plastic boxes (10×10×20 cm) on an elevated mesh floor and allowed 30 min for habituation. Tactile thresholds were measured by von Frey fibers (Stoelting, Wood Dale, IL, USA). The paw was pressed with one of a series of von Frey fibers with logarithmically incrementing stiffness (0.4, 1.0, 2.0, 4.0, 6.0, 10.0, 15.0, and 26.0 g) perpendicularly onto the plantar medial surface. Each fiber was 120
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applied for 5–6 sec. The 50% withdrawal threshold was determined using up-down method (Chaplan et al. 1994). The above protocol had been published (Gao et al. 2010; M. L. Lin et al. 2014). To test thermal thresholds, animals were put in a plastic box placed on a glass plate pre-warmed to a constant 30C (Plantar Test Apparatus, IITC, CA, USA), and the plantar surface was exposed to a beam of radiant heat underneath the glass floor. The baseline latencies were adjusted to 810 sec with a maximum of 20 sec as cut off to prevent potential heat injury. The latencies were averaged over three trials, separated by a 5-min interval (Hargreaves et al. 1988). The experimenter who performed these behavioral tests was blind to the group allocation of the rats.
Intrathecal (i.t.) administration
To evaluate the role of p-p38 in response to plantar incision and EA, the rats received intrathecal administration of a p38 inhibitor, SB203580 (Cell signaling Technology, Danvers, MA, USA). One hour before plantar incision, the rats were transiently anesthetized with high concentration of sevoflurane and back hairs were shaved to expose the lower back skin. After careful identification of L45 or L56 interspace, dural puncture to the intrathecal space was performed with a 30G needle and a Hamilton microsyringe (Hamilton Co., Nevada, USA).
Correct placement of needle was confirmed by a sign of brisk tail flick. Three doses of SB203580 (0.2, 0.5 or 2.0 mM in 10 μL) were slowly injected to the study groups. Because 139
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SB203580 had to be dissolved in dimethyl sulfoxide (DMSO) which has anti-inflammatory (Gorog and Kovacs 1968) and analgesic properties (Evans et al. 1993), two concentrations of DMSO (2% and 20%) at the identical volume were injected as vehicle control to exclude bias of DMSO's potential effect.
Immunostaining
The animals were deeply anesthetized by isoflurane for transcardial perfusion of normal saline at room temperature (RT), followed by 4C, 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The L45 spinal cord segments were carefully removed, post-fixed overnight, and cryoprotected in 30% sucrose/normal saline for 3 days. The transverse spinal sections were cut in cryostat (LEICA CM3050S, Nussloch, Germany) at a thickness of 30 μm and collected in 0.1 M PB. After blocking with 3% normal goat serum for 1 h at RT, the sections were incubated with primary antibodies containing 1% normal goat serum and 0.3% Triton X-100 overnight. Sections were then incubated with biotinylated anti-rabbit IgG (1:400, Vector Laboratories, Burlingame, CA, USA) and subsequently in an avidin-biotin-peroxidase complex/diaminiobenzidine-H2O2 solution (Elite ABC kit, Peroxidase substrate kit, Vector Laboratory). Spinal free-floating sections were mounted onto gelatin-coated glass slides, air- dried, dehydrated, cleared with xylene and coverslipped with Entellan mounting medium (Merck, Darmstadt, Germany). For double immunofluorescence, sections were incubated 158
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with a mixture of two primary antibodies from different species at 4C, followed by a mixture of Cy3-conjugated (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor 488-conjugated (1:400, Jackson ImmunoResearch) secondary antibodies at RT.
In this study, our primary antibodies include anti-phospho-p38 (rabbit, 1:400, Cell Signaling Technology), anti-neuronal nuclei (NeuN, mouse, 1:500, Millipore), anti-CD11B (OX-42, mouse, 1:50, Chemicon), and anti-glial fibrillary acidic protein (GFAP, mouse, 1:4000, Chemicon).
The stained sections were examined with Zeiss Axio Imager A2 microscopy (Göttingen, German) and images were captured with a CCD camera. At a magnification of x10, immunereactive (ir) cells in the dorsal horns were counted on randomly chosen sections, at least eight for each spinal segment from each rat, and then averaged for each rat data.
Experimental protocol
We first tested the behavioral changes in rats with plantar incision by inspecting their mechanical and heat thresholds over time (Fig. 1), then we tested effect of EA on PI-induced nociceptive hypersensitivities (Fig. 2). There were 4 groups: (1) the Naive: no treatment; (2) the PI+Sham: PI followed by repeated sham EA stimulations; (3) the PI+3 mA EA: PI with repeated 3-mA EA treatments; and (4) the PI+10 mA EA: PI with repeated 10-mA EA treatments. Behavioral tests were conducted at least 2 days before PI as baseline data and at 1 177
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h, 3 h after PI on Day 0, and 1 h after EA on Day 1, 2, and 3. Spinal p-p38 expressions on the Day 3 were compared among groups and double-staining was conducted to identify p-p38-ir cell type (Fig. 3 and 4).
To understand if EA per se induces p-p38, the naïve rats received sham EA, 2-mA EA, or 10-mA EA (Fig. 5) for 4 days were sacrificed for immunostaining analysis. In separate groups, injections of p38 inhibitor at different doses were carried out to show effects of p38 activation on incisional pain (Fig. 6). One hour before PI, 10 L of (1) saline, (2) 20%
DMSO, (3) 0.2 mM SB203580 in 2% DMSO, (4) 0.5 mM SB203580 in 5% DMSO, or (5) 2.0 mM SB203580 in 20% DMSO were i.t. injected. Behavior responses were measured at 1 h, 3 h and one day after PI.
To test the combining effect of EA and p38 inhibitor, 5 groups were included (Fig. 7):
(1) Saline: a control group of pre-PI i.t saline injection and post-PI sham needling; (2) 2%
DMSO: a control group with pre-PI i.t. 2% DMSO and post-PI sham needling; (3) 0.2 mM SB: a group with pre-PI i.t. 0.2 mM SB203580 and post-PI sham needling; (4) 3 mA EA: a group with pre-PI i.t. saline and post-PI 3-mA EA; (5) SB+EA: a group with pre-PI 0.2 mM SB203580 and post-PI 3-mA EA. Behavioral tests were perfomed at post-PI 1 h, 3 h and on the next day.
Statistical Analysis 196
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All data were presented as the mean ± S.E.M. Two-way analysis of variance (ANOVA) was conducted to analyze the influence among factors of time and treatments. Repeated- measurement ANOVA (RM-ANOVA) was conducted in the control group to assess effect of time course. One-way ANOVA with post hoc Tukey’s test was conducted to compare differences among groups. Software of PASW Statistics for Windows, version 18.0 (SPSS Inc, Chicago) was used for analysis. Statistical significance is considered when p < 0.05.
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Results
Nociceptive hypersensitivity after plantar incision
PI resulted in mechanical allodynia and heat hyperalgesia immediately after surgery and persisted for about one week (Fig. 1), a similar finding to previous studies (Ririe et al. 2003;
Wen et al. 2009). Fifty-percent mechanical thresholds significantly decreased from pre-PI baseline (BL) 23.77 0.54 g to 0.49 0.05 g at 1 h and 0.65 0.09 g at 3 h (p < 0.001 vs.
BL), remained low till Day 3 (3.61 0.41 g, p < 0.001 vs. BL), and gradually increased from D5 to D7 (13.56 0.61 g, p < 0.001 vs. BL, Fig. 1A).
Thermal hypersensitivity showed similar changes (Fig. 1B). Mean withdrawal latency before incision was 9.50 0.61 s. Significant decrease occurred at post-PI 1 h (2.02 0.24 s, p < 0.001 vs. BL) through Day 3 (4.78 0.53 s, p < 0.001) to near baseline on Day 5 (7.10 0.23 s, p = 0.101). The two tests proved incision leads to an acute, strong painful sensation which recovered rapidly within few days, as clinical patients. Therefore, we limited observation within 3 days in subsequent experiments.
High-intensity EA suppresses PI-induced mechanical hypersensitivity
EA could reduce incisional pain on the paw depending on EA stimulating intensities (Fig.
2A). High-intensity EA (10 mA) significantly reduced mechanical allodynia since the first hour post PI, and kept significantly higher thresholds than those in the sham (PI+Sham) and 221
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low-intensity EA (PI+3 mA EA) groups thereafter. Low-intensity EA (PI+3 mA EA group) did not produce any anti-allodynic effect throughout the course (p > 0.05 vs. PI+Sham).
Notably, neither 3-mA nor 10-mA EA could alter PI-induced heat hyperalgesia at any time point (Fig. 2B). Accordingly, higher strength EA is necessary to attenuate PI-induced mechanical allodynia and repeated treatment is required to maintain the effect. However, EA has no analgesic effect on heat hyperalgesia.
Repeated EA does not reduce PI-induced spinal p-p38
We found that PI drastically increased the amount of p-p38-ir cells in the spinal dorsal horn at 3 days after surgery (Fig. 3A), which spreaded diffusely without lamina differences.
However, to our astonishment, EA treatment did not decrease p-p38 expression in the dorsal horn (Fig. 3B and 3C). Quantitative analysis revealed that the 3-mA EA group did not differ from the PI+sham group, whereas the 10-mA EA group showed mildly higher p-p38-ir cell counts than those in the sham group (84.71 4.52 vs. 74.60 3.57, respectively; p = 0.049;
Fig. 3D).
p-p38 is localized within spinal microglia
We used immunofluorescent double staining to clearly identify the cells type of PI-induced p- p38. As shown in Fig. 4, a majority of p-p38 (red spots at panels A, B, C) were colocalized 240
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with OX-42-positive microglia (Fig 4B’, 4B’’). No coexpression of p-p38 with NeuN- positive neuron (Fig. 4A’, A’’) or GFAP-positive astrocyte (Fig. 4C’, C’’) could be found.
This finding is correlated with previous studies in incisional and neuropathic pain model (Ito et al. 2009; Jin et al. 2003; Wen et al. 2009).
EA per se activates spinal p38 phosphorylation
Whether EA per se induced spinal p-p38 was also examined by repeated EA stimulations in the normal rats. We found that the naïve rats (without any noxious stimulation) showed very few p-p38-ir cells in the dorsal horn (about 20/section, Fig. 5A), whereas the EA-treated rats, though without vigorous PI injury, had clear p-p38-ir cells in dorsal horns (Fig. 5B and C).
Many EA-induced p-p38-ir cells are clustered at the medial half of the superficial laminae.
All p-p38-ir cells in Fig. 5A-C, no matter in naive or 2 EA groups, show small cell bodies and small, thin, and ramified processes, a state close to a “resting” condition in comparison with those with “activated” images in Fig. 3AC. Quantitative analysis revealed that either the 3-mA or 10-mA groups could significantly increase p-p38-ir cells more than that in the naive group (both p < 0.001; Fig. 5D).
p38 inhibitor dose-dependently suppresses mechanical allodynia 259
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As predicted, p38 inhibitor effectively prevented nociceptive hypersensitivity. SB203580, when given at 1 h before PI, led to a dose-dependent suppression on mechanical allodynia for at least 3 h (3.33 0.65, 9.12 0.00, and 10.27 0.55 respectively for 0.2 mM, 0.5 mM, and 2.0 mM vs. 1.94 0.47 for saline, Fig. 6A), but all effects disappeared on the next day.
Particularly, SB203580 did not suppress heat hyperalgesia (all p > 0.05; Fig. 6B), indicating p-p38-mediated pathway may be irrelevant to heat nociception. The vehicle solvent with the highest concentration, 20% DMSO, did not have notable effect on mechanical pain but mildly inhibited heat pain (20% DMSO vs. Saline at 1 h; p = 0.014).
Low-dose p38 Inhibitor enhances low-intensity EA analgesia
Since EA per se was found to induce spinal p-p38 expression, it was deduced that inhibiting p38 activation might enhance EA analgesia. We knew that either 3-mA EA or 0.2 mM SB203580 did not produce obvious analgesia (Fig. 2A and 6A). Therefore, we hypothesized that combination of low-intensity EA and low-dose SB203580 may elicit a synergistic effect.
As expectedly, a significant enhancement of anti-allodynic effect was observed at 1 h and 3 h of Day 0 and Day 1, compared to the saline, 2% DMSO, and treated groups (all p < 0.001, Fig. 7A). Such combination showed an enhancement and prolongation of analgesia, with values greater than that of 10-mA EA (Fig. 2A) and that of 2.0 mM SB203580 (Fig. 6A). Of 277
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note, the combination did not exert any additive effect on heat hyperalgesia (p > 0.05, Fig.
7B).
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Discussion
In the present study, we demonstrated that 4-Hz EA at a high-intensity significantly reduces post-operative mechanical allodynia and repeated applications maintain analgesic effect. We also found EA at a low-intensity has no effect on incision-induced pain. More importantly, EA per se, either at a low or high intensity, activates p38 in spinal microglia in normal rats.
Co-treatment of low-intensity EA and low-dose p38 inhibitor could remarkably prolongs and enhances effect of low-intensity EA possibly via inhibiting EA-induced p38 activation.
Growing evidence proved that spinal p38 activation plays a pivotal role in pain initiation and development in neuropathic, inflammatory, or surgical pain (Ji et al. 2009; Ji and Suter 2007; Svensson et al. 2003; Wen et al. 2007; Zhuang et al. 2007). Spinal p38 activation is mainly expressed within microglia and is usually associated with microgliosis (Hanisch and Kettenmann 2007; Ji and Suter 2007). These changes start early after peripheral injuries and temporally parallel with progression of early nociceptive behaviors. Activated spinal microglia release pro-inflammatory cytokines and chemokines, further contribute to nociceptive hypersensitivities. Therefore, blockade of microglia-p38-cytokine-mediated signal pathways with agents such as p38 inhibitors, minocycline, or TNF inhibitors could reduce nociceptive processes (Ji and Suter 2007; Palladino et al. 2003; Zhuang et al. 2007).
There were studies demonstrating that EA could downregulate p-p38 or inhibit microglia in monoarthritic pain (Shan et al. 2007; Sun et al. 2008) and complete Freund's adjuvant (CFA)- 297
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induced pain (Hargreaves et al. 1988; Mi et al. 2011; K. D. Xu et al. 2010). However, whether these p-p38 reductions were due to a direct action of EA or secondary to inhibition of pre- or post-synaptic noci-responsive neurons has not yet been clarified.
A striking finding of this study is that EA per se stimulates p38 activation in the ipsilateral dorsal horns, which is in contradiction with our original hypothesis and is opposed to published studies regarding to EA effects on MAPK activations. For instance, repeated daily EA at alternating-frequencies (2 and 100 Hz) could decrease CFA-induced paw swelling, mechanical hypersensitivity, and spinal p-p38 at 3 and 14 days after CFA injection (Y. Liang et al. 2012). The authors advocated that EA owned an inhibitory effect on p-p38- mediated inflammation and hyperalgesia. Another study demonstrated that repeated low- frequent EA produced synergistic inhibition against CFA-induced heat hyperalgesia for 2 days when co-injected with dizocilpine, a NMDA antagonist (Jang et al. 2011). As EA or dizocilpine was given alone, only EA inhibited CFA-induced p38 activation. They therefore concluded that EA-mediated p-p38 inhibition is NMDA-independent. Both studies similarly indicated the importance of p-p38 in inflammatory pain and effect of EA on spinal p38 activation. Nevertheless, none of them had examined EA-evoked p-p38 activation in normal rats or co-administered p38 inhibitor for verification.
Paw incision shares many molecular reactions in common with those after CFA injection, however, discrepancies are present between them. Incision seems to result in immediate, 316
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excruciating pain much stronger than CFA-induced inflammatory pain. In this study, 3-mA EA is insufficient to reduce incision-induced allodynia, whereas low-intense EA (less than 2 mA) produced analgesia in many CFA studies (Huang et al. 2004; Jang et al. 2011; Lao et al.
2004; Y. Liang et al. 2012; R. X. Zhang et al. 2005b; Y. Zhang et al. 2011). In tail-flick and formalin-induced pain models, we demonstrated stronger EA stimulation (i.e. 20 times of muscle twitch intensity or above) led to stronger suppression, but meanwhile higher Fos expression in the spinal dorsal horns (Wen et al. 2010; Wen et al. 2007). Intense electrical stimulus sufficient to activate A and C fibers or at a pain-induction level, could also result in systemic/heterotopic analgesia in animals (Danziger et al. 2001; Morton et al. 1988; Romita and Henry 1996; Romita et al. 1997) and in humans (Roby-Brami et al. 1987; W. D. Xu et al.
2003). Collectively, strong EA stimulation may be required to suppress PI-induced strong pain, but may simultaneously trigger unwanted activations of MAPK system.
Of particular notice, we present that both 3-mA EA and 10-mA EA stimulation for 4 days activate p-p38 within spinal microglia in the normal rats, which has yet to be reported before.
Comparing to reports showing that EA below 2 mA suppressed p-p38 in CFA-induced pain, it is hypothesized that 3-mA EA strength is a boundary to determine the activation of microglial p38. When EA stimulation at strength above this level, A and C afferents may be activated to sensitize spinal noci-responsive neurons and MAPK phosphorylation. Besides, EA has been found to regulate immune response via recruitment of MAPK (p-ERK and p- 335
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p38) activations in spleenic T helper cells (K. Wang et al. 2009). On the other hand, induction of endogenous opioids and binding to -, -, and maybe -opioid receptors following EA treatment is a well-proved acupuncture analgesic mechanism (Han 2003; R. X. Zhang et al.
2004). Of many inflammatory pain conditions, low-frequent EA analgesia could be completely reversed by - or -opioid receptor antagonists or toxin for -opioid receptor (Huang et al. 2004; R. X. Zhang et al. 2004; R. X. Zhang et al. 2005a; Y. Zhang et al. 2011).
In PI model, EA analgesia was completely reversed by systemic naloxone injection (Oliveira and Prado 2000) and daily EA effect was abolished by repeated naloxone injections in our recent study (Zeng et al. 2014). Because EA-induced analgesia is intensity-dependent (Wen et al. 2010), it is thus rational to assume that "primary" opioid-dependent EA analgesia could be counteracted by concomitant recruitment of p-p38-dependent nociceptive cascades as stimulation intensity is increased. Taken together, phenotype of EA analgesia under this circumstance could be a balance between opioid-mediated anti-nociception and p-p38- pertained drawback.
The results of co-administration of EA and p38 inhibitor justify our above deduction.
When given alone, 3-mA EA and 0.2-mM SB203580 were ineffective individually. The co- administration not only magnified 3-mA EA from ineffective to strong analgesia, but also prolonged the effect to the next day. Our finding is in consistence with the concept that silencing microglial p38 activation can reduce early neuroplastic sensitization and prevent 354
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nociceptive development (Ji et al. 2013; Wen et al. 2011). Pretreatment with sufficient doses of SB203586 or FR167653, two different p38 inhibitors, produced 2-3 days of anti-allodynic effect on incision pain (Mizukoshi et al. 2013; Wen et al. 2009) and inflammatory pain (Li et al. 2010). Taken together, we believe that co-treatment could efficaciously overcome disadvantage of low analgesic potency in EA.
Here, we noted that EA did not inhibit heat hyperalgesia. Such differential analgesia for different pain patterns is possible. The anti-allodynic effect of this study is in agreement with previous PI study (Oliveira and Prado 2000) and consistent with a CFA study showing attenuation of mechanical, but not thermal hyperalgesia (Huang et al. 2004). However, there were other studies advocated that EA reduced heat hyperalgesia in CFA model (Jang et al.
2011; R. X. Zhang et al. 2005b). It is difficult to compare different results based on distinct study designs and pain models because EA actions include either inhibition or dis-inhibition of cellular signals and transmission pathways, and exert distinct functions among acute or persistent inflammatory pain (Huang et al. 2004; Lao et al. 2004; Y. Liang et al. 2012; R. X.
Zhang et al. 2005b), cancer pain (Kawasaki et al. 2008), ankle sprain pain (Koo et al. 2002), monoarthritis pain (Sun et al. 2008; Sun et al. 2006), and neuropathic pain (Dai et al. 2001;
Kim et al. 2005; Lau et al. 2008). In-depth exploration of activated mechanisms and standarization of EA protocols are required before comparing the analgesic variations.
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Conclusions
In conclusion, an inference is drawn from the present study that strong EA stimulation is necessary to produce strong analgesic effect but is possibly associated with p38 activation in spinal microglia. EA analgesia may be depressed by concomitant p38 activation. We suggested that instead of using strong EA stimulation, co-administration of low-intensity EA with p38 inhibitor may provide better chances to potentiate EA efficacy and improve pain control quality.
Conflicts of Interest
All authors declared that there is no conflict of interest in this study. Part of our results had been reported as an oral presentation form in the annual meeting of the 16th International Congress of Oriental Medicine (ICOM).
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Acknowledgments
We have especial thanks to Prof. Yi-Wen Lin (Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan) for his critical reviewing of this manuscript.
We also thank Miss Ya-Hsin Lou for her technical and official assistance. This study was sponsored by research grants from the National Science Council in Taiwan (NSC97-2314-B- 341-002-MY3, NSC101-2314-B-039-005-MY3), in part from China Medical University under the Aim for Top University Plan of the Ministry of Education in Taiwan to Y.-R. Wen, and the Joint Research Grant of Shin-Kong Wu Ho-Su Memorial Hospital and Taipei Medical University (SKH-TMU-98-15) to Y.-R. Wen and Julia Y.-R. Chen.
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Figure Legends
Figure 1: Behavioral changes after plantar incision (PI). Behavioral changes measured by the
von Frey test (A) and Hargreave’s method (B) at different time points are shown. Heat withdrawal latencies returned to pre-incision baseline within one week, but mechanical withdrawal thresholds did not. Data are presented as mean ± SEM. ### p < 0.001 v.s. pre-PI baselines (BL). Repeated measurement ANOVA followed by post hoc Tukey’s test. N = 9/group.
Figure 2: Stimulation intensity affects analgesic effect of repeated EA at ST36 (Zusanli
acupoint) on PI-induced pain. After PI surgery, rats received daily EA of 3 mA (N = 9) or 10 mA (N = 8), respectively. The naïve group (N=7) did not have any surgery or EA treatment, and the sham group (N = 9) received PI and sham needling without electrical current. EA of 10 mA attenuated paw withdrawal responses to von Frey filaments (Fig. A), but not heat withdrawal latencies (Fig. B). Data are presented as mean ± SEM. * p < 0.05, *** p< 0.001 v.s. the PI+sham group; +++ p < 0.001 v.s. the PI+3 mA EA group, and ### p < 0.001 v.s. the other three groups; one-way ANOVA followed by post hoc Tukey’s test.
Figure 3: Repeated high-intensity EA enhances PI-induced p38 activation. The sham group received PI surgery and sham needling without electrical current. Immunostaining illustrates 624
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p-p38 results in groups of the (A) PI+Sham, (B) PI+3 mA EA, and (C) PI+10 mA EA groups on Day 3. (D) Quantification of the p-p38-immunoreactive cells in the L4-5 spinal dorsal horn. N= 6/group. Data are presented as mean ± SEM. * p < 0.05 v.s. the PI+Sham group, one-way ANOVA followed by post hoc Tukey’s test. Scale bar = 50 μm.
Figure 4: PI-induced p-p38 is located within microglia in the spinal dorsal horn.
Immunofluorescent stainings of p-p38 (red, Fig. A, B, C), NeuN (A’, neuronal marker), OX- 42 (B’, microglia marker), and GFAP (C’, astrocytic marker) in the L4-5 spinal dorsal horn are shown. Double-staining demonstrates co-localization of p-p38 (red) in microglia (green) (B”, arrow heads), but not in neuron (A”) or astrocyte (C”). Scale bar = 25 μm.
Figure 5: Repeated EA stimulations induce p38 activation in the normal rats. Rats received
daily EA of 3 mA or 10 mA at right ST36 for 4 days. Immunostaining illustrates p-p38- immunoreative cells in groups of the (A) Naïve, (B) 3 mA EA, and (C) 10 mA EA. (D) Quantification of the p-p38-ir cells in the Naive group (N = 3), 3 mA EA (N = 4), and 10 mA EA (N = 4). Data are presented as mean ± SEM. *** p < 0.001 v.s. the Naïve group. One-way ANOVA followed by post hoc Tukey’s test. Scale bar = 50 μm.
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Figure 6: p38 inhibitor, SB203580, decreases PI-induced mechanical allodynia, but not heat
hyperalgesia. (A) Intrathecal SB203580 dose-dependently prevents mechanical allodynia at 1 and 3 h after PI. The effect disappears on next day; (B) SB203580 has almost no effect on heat hyperalgesia at all time points. DMSO of 20% is the concentration of vehicle for maximal SB 203580 dose (2.0 mM). N = 8 for each group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 v.s. the Saline group, and +++ < 0.001 v.s. the 0.2 mM SB group by one-way ANOVA followed by post hoc Tukey’s test.
Figure 7: Co-administration with low-dose SB203580 enhances analgesic effect of low-
intensity EA on PI-induced mechanical hypersensitivity. (A) Comparison of allodynic thresholds among the saline, the vehicle (2% DMSO), intrathecal low-dose SB203580 (0.2 mM SB), low-intensity EA (3-mA EA), and SB+EA (co-administration with 0.2-mM SB203580 and 3-mA EA) at different time points post PI. (B) Heat hyperalgesic responses among groups. N= 68 for each group. Data are presented as mean ± SEM. * p < 0.05, *** p
< 0.001 v.s. the Saline group, and +++ < 0.001 v.s. the SB+EA group by one-way ANOVA followed by post hoc Tukey’s test.
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