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Electroacupuncture Enhances the Antiallodynic and Antihyperalgesic Effects of Milnacipran in Neuropathic Rats

Li, Chengjin PhD; Ji, Byeong Uk BS; Kim, Yiquot BS; Lee, Ji Eun MS; Kim, Nam-Kwen KMD, MPH, PhD; Kim, Seung Tae KMD, PhD; Koo, Sungtae KMD, PhD

doi: 10.1213/ANE.0000000000001212
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Milnacipran, a selective serotonin/norepinephrine-reuptake inhibitor, has been shown to elicit a beneficial effect in various models of neuropathic pain. Previously, we reported that repetitive electroacupuncture (EA) significantly ameliorates neuropathic pain induced by L5 spinal nerve ligation (SNL). In the present study, we sought to determine whether a single treatment with EA produces analgesia and whether EA in combination with a subeffective dosage of milnacipran exhibits an additive effect in SNL rats.

METHODS: Mechanical allodynia and thermal hyperalgesia were assessed by measuring paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively, 1 day before and 5 days after neuropathic surgery. In addition, on day 5 post-SNL, time courses of behaviors were assessed at 0, 1, 2, 4, 6, and 8 hours after intrathecal (i.t.) milnacipran (1, 5, and 20 μg) administration. EA (10 Hz/1 mA) was administered at the ST36 and GB34 acupoints for 30 minutes on day 5 and the time courses of behaviors were also assessed at 0, 1, 2, 4, 6, and 8 hours later. Similarly, when treated in combination (milnacipran [5 μg, i.t.] and EA [10 Hz/1 mA]), time courses of behaviors were assessed at the same time points.

RESULTS: Intrathecal injection of milnacipran at 5 or 20 μg exerted dose-dependent effects on thermal hyperalgesia but had similar efficacies on mechanical allodynia. Furthermore, EA itself detectably attenuated hyperalgesia at 4 hours after the application, but no statistically significant difference was found in mechanical allodynia. Importantly, cotreatment with EA and milnacipran (5 μL) produced more potent antiallodynic and antihyperalgesic effects than those obtained from EA or milnacipran alone at 1, 2, and 4 hours after treatment, indicating an additive effect. In addition, the analgesic effect of EA plus milnacipran was almost completely abolished by the catecholamine neurotoxin 6-hydroxydopamine hydrobromide (25 μg), which depletes spinal norepinephrine, and by yohimbine (an α2-adrenoceptor antagonist, 30 μg, i.t.). Somewhat surprisingly, the analgesic effect of milnacipran plus EA lasted for 6 hours.

CONCLUSIONS: The study shows that, in male rats with SNL, spinal administration of milnacipran effectively alleviates mechanical allodynia and thermal hyperalgesia, and that a single treatment of EA has an antihyperalgesic effect. Furthermore, our findings suggest that coapplication of EA and milnacipran enhanced antiallodynia and antihyperalgesia by activating spinal noradrenergic systems coupled with spinal α2-adrenoceptors and prolongs the duration of analgesia.

Supplemental Digital Content is available in the text.Published ahead of print March 9, 2016

From the *Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, Korea; Department of Ophthalmology and Otolaryngology and Dermatology, School of Korean Medicine, Pusan National University, Yangsan, Korea; and Department of Korean Medical Science and Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan, Korea.

Accepted for publication December 29, 2015.

Published ahead of print March 9, 2016

Funding: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1043909), by the NRF grant funded by the Korea government (MSIP) (2014R1A5A2009936), and by the Research Fund Program of Research Institute for Basic Sciences, Pusan National University, Korea, 2013, Project No. RIBS-PNU-2013–104.

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

Reprints will not be available from the authors.

Address correspondence to Sungtae Koo, KMD, PhD, Department of Korean Medical Science, Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, 49 Busandaehakro, Mulgeum-eup, Yangsan, Gyeongnam, Republic of Korea 626–870. Address e-mail to stkoo@pusan.ac.kr.

Neuropathic pain resulting from nerve injury is responsible for causing persistent suffering and is characterized by spontaneous pain, allodynia, and hyperalgesia.1,2 Milnacipran is a new antidepressant and a representative serotonin/norepinephrine (NE) reuptake inhibitor, which is commonly used to treat neuropathic pain in the clinical setting.3–5 Milnacipran does not produce pharmacologically active metabolites6 and has no meaningful affinity for most receptors, such as postsynaptic muscarinic acetylcholine, opioid, histamine, or N-methyl-D-aspartate receptors, and thus, it has relatively few side effects.7–9 Furthermore, the intrathecal (i.t.) administration of milnacipran has been shown to attenuate mechanical allodynia and thermal hyperalgesia in animal models of spinal nerve ligation (SNL).10–12 Additionally, the analgesic effect of milnacipran at the spinal level appears to be mediated more by the noradrenergic system than by the serotonergic system.12

It has also been shown that many patients use complementary and alternative medicines, including acupuncture in lieu of, or as an adjunct to, conventional medicine.13 Electroacupuncture (EA) is a type of acupuncture that has been demonstrated to have analgesic effects in clinical studies.14–16 Furthermore, the antiallodynic and antihyperalgesic effects of EA have been confirmed by animal studies,17–19 and in a previous study, we showed that repetitive EA (10 Hz/1 mA for 30 minutes administered at ST36 and GB34 acupoints) significantly attenuated mechanical allodynia and thermal hyperalgesia in a rat model of neuropathic pain induced by L5 SNL.17

Nowadays, combinations of acupuncture and drugs are used for pain management and in clinical18 and well-controlled experimental settings.19–21 EA-drug combinations have been reported to be more effective at treating inflammatory pain than EA or pharmaceutical agents alone. However, few studies have investigated the analgesic effects of EA-drug combinations using a neuropathic pain model.

The present study was performed to determine whether a single treatment of EA has analgesic effects, and whether EA in combination with a subeffective dosage of milnacipran shows additive analgesic effects in a rat model of neuropathic pain.

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METHODS

Animals

Experiments were performed on young adult male Sprague-Dawley rats (200–250 g; Hyochang Science, Daegu, Korea). During the entire experimental period, animals were housed in pairs in plastic cages with soft bedding under a 12/12-hour reversed light-dark cycle (dark cycle: 8:00 AM–8:00 PM) and temperature (22–25°C). Food and water were available ad libitum. All experimental procedures were performed in accordance with the Animals (Scientific Procedures) Act 2008 (Korea) and all complied with the recommendations of the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. The study was also approved by the Ethics Committee on Animal Research at Pusan National University (PNU-2014-0602).

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Surgery for Intrathecal Catheterization

Intrathecal catheterization was performed using polyethylene tubing (PE-10, outer diameter: 0.61 mm, inner diameter: 0.28 mm; Becton Dickinson, Sparks, MD). Rats were anesthetized with isoflurane (3% induction, 2% maintenance) in a N2O/O2 mix, and then an 8-cm-long PE-10 tube was inserted caudally into the subarachnoid space of the spinal lumbar enlargement through the atlanto-occipital membrane.22 The catheter was secured to the musculature and muscles, and skin were sutured in layers. After catheterization, rats were returned to their home cages and allowed to recover before neuropathic surgery. Animals showing signs of motor dysfunction were excluded.

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Drug Administration

Milnacipran hydrochloride (Pierre Fabre Médicament, Boulogne, France) and the α2-adrenoceptor antagonist, yohimbine hydrochloride (#Y3125; Sigma, St. Louis, MO), were dissolved in isotonic saline solution (0.9% NaCl) to concentrations of 1, 5, and 20 μg/10 μL and 30 μg/10 μL, respectively.

The neurotoxin, 6-hydroxydopamine hydrobromide (6-OHDA, H116; Sigma), primarily causes lesions in noradrenergic neurons.23 The 6-OHDA solution used contained 10 mM 6-OHDA and 0.01% (w/v) ascorbic acid. 6-OHDA (25 μg) or 0.9% NaCl containing 0.1 mg/mL ascorbic acid (vehicle) was administered immediately after i.t. catheterization, that is, 7 days before SNL.

All drugs were delivered in a volume of 10 μL and administered intrathecally within 30 seconds. Injections were followed by a 10-μL saline flush.

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Experimental Design

The study involved 5 series of experiments. In the first, the dose-response effects of i.t. milnacipran (1, 5, and 20 μg) were examined. In the second, additive analgesic effects of EA combined with milnacipran was investigated. In the third, motor function was assessed to determine the effects of 6-OHDA pretreatment. In the fourth and fifth, we used a chemical sympathectomy method24 using 6-OHDA and performed antagonist studies with yohimbine to probe the antinociceptive mechanism of milnacipran and EA in combination.

Animals were divided into 11 groups of 4 to 10 rats per group: (1) SNL + Sal, (2) SNL + Mil (1 μg), (3) SNL + Mil (5 μg), (4) SNL + Mil (20 μg), (5) SNL + EA + Sal, (6) SNL + EA + Mil (5 μg), (7) SNL + Yoh (30 μg), (8) SNL + Yoh (30 μg) + EA + Mil (5 μg), (9) ascorbic + SNL + EA + Mil (5 μg), (10) 6-OHDA (25 μg) + SNL + EA + Mil (5 μg), and (11) 6-OHDA (25 μg) + SNL + Sal. Since we could not handle such a large number of animals simultaneously, repeated experiments were conducted to collect the data, and thus we allocated the animals into different groups at random.

Figure 1

Figure 1

On day 5 post-SNL, time courses of behaviors were investigated at 0, 1, 2, 4, 6, and 8 hours after i.t. milnacipran. The protocols of the fourth and fifth series of experiments are provided in Figure 1.

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EA Treatment

EA was administered for 30 minutes at 10 Hz and 1 mA with a 1-millisecond pulse width. In detail, EA was applied under gaseous anesthesia (3% for induction and 1.5% for maintenance in a mixed N2O/O2) by stimulating acupuncture points using a pair of bipolar electrodes (modified acupuncture needles). A stainless steel acupuncture needle (0.25-mm diameter and 40-mm long) was mounted on a holder and inserted into a specific point at a depth of 5 mm. Stimulation was applied using a Pulsemaster Multi-channel Stimulator SYS-A 300 (World Precision Instruments, Inc., Berlin, Germany).

EA was applied to 2 different points, which were equivalent to specific human acupuncture points on ipsilateral sides (right) to surgically treated hind limbs. The first point was the Joksamli point (ST36), which is located at the anterior aspect of the leg, lateral to the tibial tubercle at the midpoint of the anterior tibialis.25 The second point used was the Yangneungcheon point (GB34), which is located on the fibular aspect of the leg, in the depression anterior and distal to the head of the fibula.25 These 2 acupoints were chosen based on the results of our previous study17 because EA at these 2 points was found to exert a strong analgesic effect on neuropathic pain.

Immediately after the termination of EA, milnacipran or 0.9% NaCl was administered intrathecally.

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Neuropathic Surgery: SNL Models

Rats were anesthetized with isoflurane (3% induction and 2% maintenance) in N2O/O2. A midline skin incision was then made on the back at the lower lumbar region, the paraspinal muscles retracted, and the left transverse process of the L6 vertebra removed under a dissection microscope. The right L5 spinal nerve was then identified, gently separated from the adjacent L4 spinal nerve, and tightly ligated using 6-0 silk thread, as described in a previous report.2

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Evaluation of Neuropathic Pain Sensitivity

Mechanical Allodynia

Mechanical allodynia was assessed by measuring the threshold level required to induce hind paw withdrawal from a graded force applied to the plantar hind paw surface with a von Frey filament using a Dynamic Plantar Aesthesiometer (Ugo Basile, Varese, Italy). The cutoff force was set at 40 g to prevent potential tissue damage. A reduction in paw withdrawal threshold (PWT) after SNL was considered as sign of mechanical allodynia.

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Thermal Hyperalgesia

Thermal hyperalgesia was assessed by measuring the time taken (latency) to withdraw a hind paw from a focused beam of radiant heat (infrared intensity was 70) applied to the plantar surface using a commercial plantar test unit (Ugo Basile). A cutoff latency of 20 seconds was imposed on nonresponsive animals. A significant reduction in paw withdrawal latency (PWL) after SNL was considered as a sign of thermal hyperalgesia.

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Assessment of Motor Function

Because motor impairment can affect an animal’s ability to respond to noxious stimuli, a rotarod treadmill (LE8500; Panlab-Harvard Apparatus, Barcelona, Spain) was used to determine whether 6-OHDA treatment induced any changes in motor performance. The rotarod test was performed 7 days after administering 6-OHDA. After a training period, animals were placed on the rod, which was accelerated from 4 to 40 rpm over 5 minutes. Total residence time on the rod was recorded automatically.

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Statistical Analyses

All results are presented as means ± SEMs. To determine the significance of differences between behavioral scores before and after milnacipran and/or EA treatment, if both normality test (Shapiro-Wilk) and equal variance test (Levene) passed, 1-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni t test (multiple comparisons versus control group) was used, and if either normality or equal variance test failed, Friedman repeated-measures ANOVA on ranks was used (Supplemental Digital Content, http://links.lww.com/AA/B371). The paired t test was used to determine the differences between behavioral scores before and after 6-OHDA treatment. The Student t test was used to determine the differences between rotarod residence time in the 6-OHDA and vehicle (ascorbic acid)-pretreated groups. In addition, to determine the significance of intergroup differences, if both the normality test (Shapiro-Wilk) and the equal variance test (Levene) were passed, 1-way ANOVA followed by Tukey test was used. If either the normality or the equal variance test failed, Kruskal-Wallis ANOVA followed by Dunn method was used (Supplemental Digital Content, http://links.lww.com/AA/B371). Statistical analysis was performed using SigmaPlot program version 12.0 (Systat Software Inc., San Jose, CA). Statistical significance was accepted for P values <0.05.

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RESULTS

The Effect of Milnacipran on Neuropathic Pain Symptoms

Before SNL, no significant intergroup differences were observed in overall mean baseline PWT and PWL to mechanical and noxious heat stimuli, but after SNL, PWT and PWL both decreased.

As shown in Figure 2, PWLs in the SNL + Mil (20 μg) group at 1 hour (#P < 0.001) and 2 hours (#P = 0.039) were higher than basal PWLs (0 hour) measured before milnacipran administration (1-way repeated ANOVA followed by Bonferroni test [multiple comparisons versus control group]; Fig. 2B) and higher than those in the SNL + Sal at 1 hour (@Q = 3.617, Kruskal-Wallis 1-way ANOVA on ranks was followed by Dunn method (Fig. 2B) and 2 hours (@P = 0.034, 1-way ANOVA followed by Tukey test). In addition, PWLs in the SNL + Mil (5 μg) group at any appointed time points showed no statistically significant difference versus basal PWLs (0 hour) (P = 0.051, Friedman repeated-measures ANOVA on ranks), but the P value was 0.051, and PWLs in the SNL + Mil (5 μg) group showed a pattern similar to those in the SNL + Mil (20 μg) group. Moreover, significant differences in PWTs were observed before to 1 hour (*P < 0.001) and 2 hours (*P = 0.015) after milnacipran administration in the SNL + Mil (5 μg) group and before to 1 hour (#P < 0.001) in the SNL + Mil (20 μg) group (1-way repeated ANOVA followed by Bonferroni test [multiple comparisons versus control group]; Fig. 2A).

Figure 2

Figure 2

These results show that milnacipran (5 or 20 μg, i.t.) produced a dose-dependent effect on thermal hyperalgesia, but there was no dose-dependent effect in mechanical allodynia. At 1 μg, milnacipran had limited effects on mechanical allodynia and thermal hyperalgesia. Peak effects were achieved at 1 hour after administration.

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The Effects of EA, Milnacipran, and EA Plus Milnacipran on Symptoms of Neuropathic Pain

Figure 3

Figure 3

As shown in Figure 3, in the SNL + EA + Sal group, PWLs were higher than basal PWLs (0 hour) at 4 hours (!P < 0.001, 1-way repeated ANOVA followed by Bonferroni test [multiple comparisons versus control group]), and higher than those in the SNL + Sal (&P < 0.001) and SNL + Mil (5 μg) (%P = 0.002) and SNL + Mil (20 μg) (^P < 0.001) groups at 4 hours postadministration (1-way ANOVA followed by Tukey test; Fig. 3B). Furthermore, PWLs in the SNL + EA + Sal group were also higher than those in SNL + Mil (20 μg) group at 1 hour time point (^Q = 3.062, Kruskal-Wallis 1-way ANOVA on ranks followed by Dunn Method; Fig. 3B), but EA did not have any effect on PWTs (Fig. 3A). In the SNL + EA + Mil (5 μg) group, PWTs were greater at 1 hour (*P = 0.031) and 4 hours (*P = 0.022), and PWLs were greater at 4 hours (*P = 0.033) postadministration than basal PWTs and PWLs (0 hour), respectively (1-way repeated ANOVA followed by Bonferroni test [multiple comparisons versus control group]); Fig. 3A). In addition, regarding mechanical allodynia (Fig. 3A), SNL + EA + Mil (5 μg) had a more potent effect than SNL + Sal at 1 hour (#P = 0.044) and 2 hours (#P = 0.023), and SNL + EA + Sal at 1 hour (@P = 0.016). The intergroup analyses were performed using 1-way ANOVA followed by Tukey test. Although there was no statistically significant difference between different groups at 4-hour time point, the P value was 0.054 (Kruskal-Wallis 1-way ANOVA on ranks). Regarding thermal hyperalgesia (Fig. 3B), the SNL + EA + Mil (5 μg) was more potent than SNL + Sal at 4 hours (#P < 0.001) and 6 hours (#P = 0.044), SNL + Mil (5 μg) at 4 hours (+P < 0.001), and SNL + Mil (20 μg) at 4 hours ($P < 0.001). The intergroup analyses were performed using 1-way ANOVA followed by Tukey test. The additive analgesic effects of EA combined with milnacipran lasted 6 hours.

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The Effects of 6-OHDA on Motor Function

Figure 4

Figure 4

As shown by the results presented in Figure 4, the Student t test revealed no significant difference between 6-OHDA-pretreated and vehicle (ascorbic acid)-treated rats (P = 0.302, Student t test, n = 9 per group) with respect to motor function, indicating that 6-OHDA pretreatment did not affect motor function.

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The Effects of 6-OHDA Pretreatment on Additive Analgesic Effects of EA Plus Milnacipran on Symptoms of Neuropathic Pain

To examine the involvement of spinal noradrenergic systems in the analgesic effect of EA plus milnacipran, spinal noradrenergic neurons were selectively denervated using 6-OHDA.

Figure 5

Figure 5

As shown in Figure 5, no behavioral alterations were observed among ascorbic + SNL + EA + Mil (5 μg), 6-OHDA (25 μg) + SNL + EA + Mil (5 μg), and 6-OHDA (25 μg) + SNL + Sal groups, when tested 7 days after 6-OHDA pretreatment (6-OHDA) and 5 days post-SNL (0 hour). However, the ascorbic + SNL + EA + Mil (5 μg) exhibited a significant antiallodynic effect at 1 (*P = 0.027) and 4 hours (*P = 0.004) postadministration, and a significant antihyperalgesic effect at 1 hour (*P < 0.001), 2 hours (*P = 0.026), 4 hours (*P = 0.019), and 6 hours (*P = 0.045) versus PWTs and PWLs at 0 hour (1-way repeated ANOVA followed by Bonferroni test [multiple comparisons versus control group]). However, pretreatment with 6-OHDA significantly blocked mechanical allodynia at 4 hours postadministration (#P = 0.036) and heat hyperalgesia at 1 hour (#Q = 2.745), 2 hours (#Q = 3.145), and 6 hours (#P = 0.019) compared with ascorbic + SNL + EA + Mil (5 μg) (1-way ANOVA followed by Tukey test or Kruskal-Wallis 1-way ANOVA on ranks followed by Dunn method). In addition, regarding mechanical allodynia, though there was no statistically significant difference between 3 groups at 6-hour time point, the P value is 0.053 (1-way ANOVA).

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The Effects of Yohimbine Pretreatment on the Antinociceptive Effects of EA Plus Milnacipran on Symptoms of Neuropathic Pain

Figure 6

Figure 6

To evaluate the possible involvement of α2-adrenoceptors in the additive analgesic effects shown by EA plus milnacipran, a yohimbine blockade experiment was conducted. As shown in Figure 6, SNL + EA + Mil (5 μg) increased PWTs at 1 hour (*P = 0.031) and 4 hours (*P = 0.022) postadministration versus PWTs at 0 hour and increased PWLs at 4 hours (*P = 0.033) versus PWLs at 0 hour (1-way repeated ANOVA followed by Bonferroni test [multiple comparisons versus control group]). However, yohimbine pretreatment almost completely blocked the inhibitory effect of EA plus milnacipran on mechanical allodynia at 1 hour (#P = 0.003) and 4 hours (#P = 0.022) and thermal hyperalgesia at 4 hours (#P < 0.001), SNL + EA + Mil (5 μg) vs SNL + Yoh (30 μg) + EA + Mil (5 μg) group; +P = 0.01 (mechanical, 1 hour), +P = 0.002 (thermal, 4 hours), SNL + EA + Mil (5 μg) vs SNL + Yoh (30 μg) group, 1-way ANOVA followed by Tukey test. In addition, regarding mechanical allodynia, though there was no statistically significant difference between 3 groups at 2-hour time point, the P value is 0.019 (Kruskal-Wallis 1-way ANOVA on ranks followed by Dunn method).

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DISCUSSION

We selected the SNL model because it is one of the most commonly used animal models of neuropathic pain. Furthermore, nerve injury–induced hypersensitivity is believed to result from changes in the peripheral and central nervous systems,26–28 and SNL can induce nerve injury–induced hypersensitivity.29 In addition, robust tactile and thermal hypersensitivity occurs within 3 days of surgery and lasts for several weeks,2 and thus we conducted the time course experiments on day 5 post-SNL, after neuropathic pain developed.

Most antidepressants can produce analgesic effects at supraspinal,30 spinal,31 and peripheral sites.32 However, when antidepressants are administered systemically, it is difficult to determine the sites responsible for antinociceptive effects. Furthermore, the antiallodynic and antihyperalgesic effects of milnacipran on neuropathic pain induced by SNL are principally mediated at supraspinal and spinal sites via activation of the spinal noradrenergic system.12 Thus, we injected all drugs intrathecally to target the active site, namely, the spinal cord. Although drug effects at brain or dorsal root ganglia cannot be excluded, drugs mainly act at the spinal level after i.t. injection.22

The doses of milnacipran used in the study were chosen based on the findings of the previous studies. Previously determined antiallodynic doses of milnacipran were 10 μg/5 μL10; 10, 30, and 100 μg in male rats with SNL11; 7 and 21 μg in male mice with SNL12; 0.3 and 1 μg in female rats with SNL33; 10−6, 10−5, 10−4, 10−3, 10−2, and 10−1 M in male rats with chronic constriction injury; and 10−3, 10−2, and 10−1 M in streptozotocin-induced diabetic rats.34 In this study, we first selected doses (0.1 and 1 μg/10 μL), at which the combination of EA and milnacipran did not exhibit an additive effect (data not shown), and subsequently, we used doses of 5 μg/10 μL and 20 μg/10 μL, at which milnacipran had a dose-dependent response, and found that the combination of EA and milnacipran at a subdose of 5 μg/10 μL produced an additive analgesic effect. Differences between studies regarding the effective dose of milnacipran are probably due to the considerable difference in animal models and species, genders, and measurement techniques used.

In a previous report, 1 EA treatment significantly reduced mechanical allodynia.35,36 However, our results show that a single EA treatment significantly decreased thermal hyperalgesia, but not mechanical allodynia. We believe that this may be due to the methodological differences in administering EA, selected neuropathic pain models, and pain assessment. In addition, our results demonstrate that nerve injury–induced thermal and tactile hypersensitivities are mechanistically distinct.26 Nerve injury–induced thermal hypersensitivity is believed to be mediated by the abnormal activities of C fibers, whereas tactile hypersensitivity is mediated by A-β fibers.37,38 Thus, the decrease in thermal hyperalgesia elicited by EA was possibly caused by changes in the activities of C fibers.

Because the blood-brain barrier is impermeable to 6-OHDA,39 NE-neurons were lesioned by administering 6-OHDA intrathecally. We injected 25 μg of 6-OHDA in a volume of 10 μL 7 days before SNL. It has been previously reported that 20 μg of 6-OHDA delivered intrathecally depletes NE levels by >85% in the spinal cord 7 days later without affecting serotonin or dopamine levels.40–42 The present study is limited by a lack of quantification of monoamine depletion by high performance liquid chromatography, and thus, we cannot exclude the involvement of spinal dopamine systems to the effect of 6-OHDA. Interestingly, descending dopaminergic regulation of neuropathic hypersensitivity responses also involves α2-adrenoceptors.43 6-OHDA does not impair the motor or sensory functions of animals, which, in the present study, were confirmed by unchanged rotarod test, PWT, and PWL results. Furthermore, these results are in line with reports of no change in tail flick and hot plate test nociceptive latencies after pretreatment with 6-OHDA.41,44 The brain-spinal descending NE inhibitory system may play important roles in neuropathic pain states.45,46 The activation of spinal adrenoceptors has been reported to produce antinociceptive effects in various rodent pain models.46,47 Furthermore, α2-adrenergic receptors, but not α1-adrenergic receptors, in the spinal cord, have been implicated in the antinociceptive effect of NE on neuropathic pain,48 which is why antagonism of the enhanced analgesic effects of EA combined with milnacipran by yohimbine (an α2-adrenoceptors antagonist) is thought to probe the antinociceptive mechanism. Moreover, other authors have suggested that the analgesic effect of milnacipran may be dominantly mediated by the noradrenergic system rather than by the serotonergic system at the spinal level.12,34 And antiallodynic effect of milnacipran, attenuated by i.t. coadministration of yohimbine, has been reported.11 Furthermore, EA antinociception might also be due to the activations of noradrenergic and serotonergic descending pain-inhibitory pathways.49,50 Additionally, EA antinociception may also occur by release of endogenous opiates.51 And, most importantly, when combined with the selective NE inhibitor, maprotiline, the antinociceptive intensity and duration of morphine were both increased. Interestingly, when pretreated by yohimbine, the area under curve yielded by the combination was decreased by about 86%.52 The above studies indicate that the additive analgesic effect of milnacipran plus EA may be related to descending noradrenergic systems.

In addition, upon termination of NE synaptic transmission, NE is reuptaken or metabolized to 4-hydroxy-3-methoxyphenylglycol (MHPG), and an elevated MHPG/NE ratio is considered to reflect increased NE turnover and, therefore, activation of the descending noradrenergic system.53,54 In the present study, the mechanisms responsible for the additive analgesic effects of milnacipran and EA were shown by the depletion of spinal NE after neurotoxin and pharmacological blockade of spinal α2-adrenoceptors by yohimbine. In future studies, spinal NE and its major MHPG contents will be measured using methods previously reported.55

It has been reported that ketamine potentiates the effect of EA on mechanical allodynia in rats with SNL.36 Moreover, EA and cell therapy have been shown to synergistically attenuate hyperalgesia induced by chronic constriction injury in rats,56 and the i.t. administration of subeffective doses of milnacipran was found to significantly enhance the spinal cord stimulation–induced suppression of mechanical hypersensitivity induced by sciatic nerve ligation in rats.57 These reports support the potential clinical applications of EA and milnacipran.

In conclusion, our results show that, in male rats with SNL, spinal administration of milnacipran effectively alleviates SNL-induced mechanical allodynia and thermal hyperalgesia. Furthermore, a single treatment of EA reduced thermal hyperalgesia but not mechanical allodynia. Importantly, the combination of a subeffective dose of milnacipran (5 μg) and EA produced greater antiallodynic and antihyperalgesic effects than either treatment alone. Finally, we suggest the descending noradrenergic pathway coupled with spinal α2-adrenoceptors might mediate the additive ameliorative effects of the EA/milnacipran combination.

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DISCLOSURES

Name: Chengjin Li, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Chengjin Li has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Byeong Uk Ji, BS.

Contribution: This author helped conduct the study.

Attestation: Byeong Uk Ji has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Yiquot Kim, BS.

Contribution: This author helped conduct the study.

Attestation: Yiquot Kim has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ji Eun Lee, MS.

Contribution: This author helped conduct the study.

Attestation: Ji Eun Lee has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Nam-Kwen Kim, KMD, MPH, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Nam-Kwen Kim has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Seung Tae Kim, KMD, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Seung Tae Kim has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Sungtae Koo, KMD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Sungtae Koo has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Jianren Mao, MD, PhD.

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REFERENCES

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2. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain. 1992;50:355–63
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