Neurochem Res (2010) 35:1780–1786 DOI 10.1007/s11064-010-0244-x

ORIGINAL PAPER

Synergetic Analgesia of Propentofylline and Electroacupuncture by Interrupting Spinal Glial Function in Rats Ling-Li Liang • Jia-Le Yang • Ning Lu¨ • Xi-Yao Gu • Yu-Qiu Zhang • Zhi-Qi Zhao

Accepted: 3 August 2010 / Published online: 18 August 2010 Ó Springer Science+Business Media, LLC 2010

Abstract Previous studies indicated that disruption of glial function in the spinal cord enhanced electroacupuncture (EA) analgesia in arthritic rats, suggesting glia is involved in processing EA analgesia. To probe into the potential value for clinical practice, the present study was to investigate the effect of propentofylline, a glia inhibitor, on EA analgesia in rats. Mechanical allodynia induced by tetanic stimulation of sciatic nerve (TSS) was used as a pain model. On day 7 after TSS, EA treatment induced a significant increase in paw withdrawal threshold to mechanical stimulation. Intrathecal or intraperitoneal injection of propentofylline relieved TSS-induced mechanical allodynia. The combination of low dosage of propentofylline and EA produced more potent anti-allodynia than propentofylline or EA alone. Immunohistochemistry exhibited that TSSinduced activation of microglia and astrocytes was inhibited significantly by propentofylline. These results indicate that propentofylline and EA induce synergetic analgesia by interrupting spinal glial function. Keywords Electroacupuncture
Propentofylline
Microglia
Astrocytes
Tetanic stimulation of sciatic nerve

Ling-Li Liang, Jia-Le Yang contributed equally. L.-L. Liang
J.-L. Yang
N. Lu¨
X.-Y. Gu
Y.-Q. Zhang
Z.-Q. Zhao (&) Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Room 1201, Ming-Dao Building, 138 Yi-Xue-Yuan Road, 200032 Shanghai, China e-mail: [email protected]

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Introduction Peripheral nerve injury often leads to neuropathic pain, which is characterized by spontaneous pain, allodynia and hyperalgesia. Numerous studies have explored pathological mechanisms underlying neuropathic pain [1–3]. Although many drugs and techniques for treatment of pain have been applied to clinical practice based on these pathogenetic mechanisms, there are variable and some side effects [2]. Acupuncture has been used for healing various diseases in traditional Chinese medicine for more than 2,000 years. Electroacupuncture (EA), a modified acupuncture technique, has been widely used for analgesia in human subjects and experimental animals with neuropathic pain [4–8]. Increasing evidence has revealed that microglia and astrocytes in the spinal dorsal horn is involved in the induction and maintenance of pathological pain [9, 10]. A variety of pathological changes such as peripheral inflammation, neuropathy and bone cancer intensely evoke activation of spinal glial cells strengthening pain [11–14]. It has been reported that inhibition of microglia attenuated inflammation-induced mechanical allodynia in rats [15] and enhanced EA analgesia in inflammatory pain rats [16, 17]. However, whether disruption of glial function enhances EA analgesia in neuropathic pain conditions has not been addressed. Propentofylline, a methylxanthine derivative, has been used in clinical trials for treatment of Alzheimer disease and vascular dementia [18, 19]. Likewise, this compound attenuates microglial and astrocytic activation and mechanical allodynia in a rodent neuropathic pain model [14]. Our previous study showed that tetanic stimulation of sciatic nerve (TSS) induced activation of glial cells and a long-lasting mechanical allodynia [3, 20]. Our recent data also showed that such tetanic stimulation elicited injury of the sciatic nerve, indicating that tetanic stimulation-induced pain

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characterizes as neuropathic pain [21]. Using this pain model, the present study was to examine synergetic analgesic action of propentofylline and EA via modulating glial function.

Experimental Procedure Animals Experiments were performed on adult male Sprague–Dawley rats (Shanghai Laboratory Animal Center, Shanghai Institutes for Biological Sciences, China) weighing 250–350 g. Rats were housed in plastic cages, four per cage. Rats were maintained on a 12:12 h light–dark cycle and a constant room temperature of 25°C, with food and water ad libitum. All experiments were carried out in accordance with the Animal Care and Use Committee of Fudan University and were complied with recommendations of the International Association for the Study of Pain. All efforts were made to minimize pain or discomfort of animals. Surgical Procedures for Tetanic Stimulation of Sciatic Nerve (TSS) and Drug Administration via Lumbar Puncture The rats were anesthetized with sodium pentobarbital (80 mg/kg, i.p.). A pair of silver electrode hook was inserted under the nerve followed by exposition of left sciatic nerve at mid-thigh level. The hindleg and pelvis were fixed with hands to avoid nerve contact with muscle caused by muscle contraction when stimulating the nerve. The stimulation parameter was according to that used in our previous electrophysiological study [22] which was consisted of 10 trains of 2 s duration with 10 s intervals, 100 Hz, 500 ls rectangular pulses at 40 V. The stimulation lasted for 1 min and 30-s. The muscle and skin layers were then sutured. The sciatic nerves of sham-operated rats were identically exposed and manipulated, but not stimulated. On day 7 after TSS, propentofylline (PPF; Sigma– Aldrich, St Louis, MO, USA) were administered by lumbar puncture (l.p.) or intraperitoneal injection (i.p., 1 mg/kg). Three dosages of propentofylline at 0.1 lg (8.16 lM), 1 lg (81.6 lM), or 10 lg (816 lM) dissolved in 20 ll of normal saline (NS) were delivered via lumbar puncture. Rats were anesthetized with 2% isoflurane and then injected at the L5-L6 interspace using a needle (0.4 mm) connected to a 250 ll syringe with 20 ll solution. EA Treatment The unilateral EA treatment was executed as previously described [17]. Rats were loosely immobilized in a

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specially made restrainer. Two stainless steel needles of 0.3 mm diameter were inserted into two acupoint, one is ‘‘Huantiao’’ (GB30, located the lateral 1/3 and medial 2/3 of the distance between the sacral hiatus and the greater trochanter of the femur), and the other is ‘‘Yanglingquan’’ (GB34, located in the depression anterior and inferior to the fibula capitulum). Constant current square-wave electric stimulation generated by H.A.N.S. Acupuncture point Nerve Stimulator (LH-202H, Huawei, Beijing, China). The intensity of the stimulation was increased stepwise from 1 to 2 mA, and then to 3 mA, with each step lasting for 10 min, totaling 30 min; at a 2 and 100 Hz alternating frequencies (automatically shifting between 100 and 2 Hz stimulation for 3 s each). Sham EA animals receiving needle insertion into GB30 and GB34 without electric stimulation were used as control. Von Frey Test for Mechanical Allodynia Mechanical withdrawal threshold was assessed by von Frey filaments (Stoelting, Wood Dale, IL, USA) as previously described [11]. All rats were acclimated to the testing procedure before testing. A series of nine calibrated von Frey filaments were applied to the central region of the plantar surface of one hind paw in ascending order (1, 1.4, 2, 4, 6, 8, 10 and 15 g) with the highest filament at 15 g. A trial consisted of application of a von Frey filament to the hind paw five times at 15 s intervals. When the hind paw was withdrawn from a particular filament in four out of the five consecutive applications, the value of that filament in grams was considered to be the paw withdrawal threshold (PWT). All the testing for detecting analgesic effects of EA and/ or propentofylline were conducted on day 7 after TSS and were done in a blinded fashion. Data were converted to percentage of maximal possible effect (%MPE) using the following formula: %MPE = (PWTpost-treatment PWTpre-treatment)/(PWTcutoff value - PWTpre-treatment) 9 100. The EA was considered to be valid only when MPE was more than 20%. In each group, the ratio of the responsive rats to total rats was calculated and defined as the analgesic ratio (%). Immunohistochemistry The rats were deeply anesthetized with an overdose of chloral hydrate anesthesia (500 mg/kg, i.p.) and perfused intracardially with NS (37°C) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 4°C). The spinal cord at L4/5 segments were dissected out and transversely cut (35 lm) in a cryostat (CM1900, Leica, Wetzlar, Germany) and processed for immunofluorescence.

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Ionized calcium-binding adaptor molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) were used as the markers for microglia and astrocytes in the spinal cord, respectively. The sections were blocked with 10% donkey serum in 0.01 M phosphate buffered saline (PBS) with 0.3% Triton X-100 for 2 h at room temperature and were incubated overnight at 4°C with rabbit anti-Iba-1 (1:2000; WAKO, Osaka, Japan) or mouse anti-GFAP (1:2000; Sigma–Aldrich, St Louis, MO, USA). Following four 10 min rinses in 0.01 M PBS, the sections were then incubated for 2 h at 4°C with rhodamine- or FITC -conjugated secondary antibody (1:200, Jackson Immunolab, West Grove, PA, USA). After coverslipped with a mixture of 50% glycerin in 0.01 M PBS, spinal cord sections were observed with the fluorescence microscope (DMRXA, Leica) and images were captured with a CCD spot camera. The entire spinal dorsal horn was taken at 109 magnification. The optical density of immunoreactive staining for Iba-1 or GFAP per section was measured in the spinal dorsal horn using a computerized image analysis system (Adobe Photoshop CS3, Adobe, San Jose, CA, USA). Three to five rats were selected from every group at each dosage. For each animal, six to eight sections of spinal cord were randomly selected for quantitative evaluation. Statistical Analysis SigmaStat 3.1 software was used to perform the statistical analysis. Statistical significance for behavioral testing was analyzed with two-way ANOVA (treatment 9 time) followed by Holm-Sidak post hoc test. Statistical significance for immunohistochemistry results was analyzed with oneway ANOVA followed by Holm-Sidak post hoc test. The results were presented as mean ± standard error of mean (SEM). A probability value less than 0.05 was considered statistically significant.

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Fig. 1 Mechanical allodynia induced by tetanic stimulation of sciatic nerve (TSS) in rats. Paw withdrawal threshold (PWT) was used to assess mechanical allodynia. Measurements are expressed as mean ± SEM. ** P \ 0.01, *** P \ 0.001, versus the sham group, two-way ANOVA followed by Holm-Sidak test, n = 13 in the sham group, n = 72 in the TSS group

TSS-operated rats on 2, 4 and 7 days after TSS, compared with the sham group (F1, 310 = 49.611, P \ 0.001). Analgesic Effect of EA on Tetanic Stimulation-induced Mechanical Allodynia The effect of EA on mechanical allodynia induced by TSS was illustrated in Fig. 2a. Following application of EA to ‘‘Huantiao’’ (GB30) and ‘‘Yanglingquan’’ (GB34) acupoints (2/100 Hz alternation, 1-2-3 mA, 30 min) on 7 days after TSS, mechanical allodynia was remarkably relieved, compared with the sham EA group (F1, 48 = 7.659, P \ 0.01). The percentage of maximal possible effect (%MPE) of EA was used for evaluating analgesic effects. MPE were increased significantly at 1 h, but not at 2 or 3 h after EA treatment, whereas MPE showed little change after sham EA treatment. Analgesic Effect of Propentofylline on Tetanic Stimulation-Induced Mechanical Allodynia

Results Mechanical Allodynia Induced by Tetanic Stimulation of the Sciatic Nerve Mechanical allodynia was assessed by measuring the paw withdrawal threshold (PWT) to von Frey filaments on 2, 4 and 7 days after tetanic stimulation of the sciatic nerve (TSS) in rats. Consistent with our previous data [21], TSS induced long-lasting mechanical allodynia in rats. As shown in Fig. 1, the PWTs in the sham group did not show any significant change on all detected days after surgery. However, the ipsilateral PWTs decreased significantly in

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The effect of propentofylline intrathecally administered via lumbar puncture at different dosage on mechanical allodynia induced by TSS was illustrated in Fig. 2b. On day 7 after TSS, pain thresholds were measured at 1, 2, 3, 4, 5, 6 and 24 h after intrathecal injection of propentofylline or NS (Fig. 2b). Propentofylline (0.1, 1 and 10 lg, l.p.) dose-dependently relieved mechanical allodynia induced by TSS. Propentofylline at the dosage of 1 and 10 lg, but not 0.1 lg, significantly relieved tetanic stimulationinduced mechanical allodynia, compared with the NS group (F3, 126 = 33.260, P \ 0.001, 10 or 1 lg propentofylline group vs. NS group). The inhibitory effect on

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Fig. 2 Analgesic effects of electroacupuncture (EA), propentofylline (PPF) or combination of both on mechanical allodynia induced by tetanic stimulation of sciatic nerve (TSS). All behavior tests were determined on day 7 after TSS. EA treatment was given 2 h after intrathecally (l.p.; b–d) or intrapertoneally (i.p., 1 mg/kg; e, f) PPF administration when using combined therapy. EA treatment was given in the ipsilateral ‘‘Huantiao’’ (GB30) and ‘‘Yanglingquan’’ (GB34) acupoints (100/2 Hz alternation, 1-2-3 mA, 30 min). %MPE was used as index to evaluate analgesic efficacy. Measurements are expressed as mean ± SEM. Statistical significance was analyzed by two-way ANOVA followed by Holm-Sidak test. a ** P \ 0.01, versus the sham EA group. b * P \ 0.05, *** P \ 0.001, versus the NS group. c, d * P \ 0.05, versus the EA group. e &&& P \ 0.001, versus baseline, * P \ 0.05, ## P \ 0.01 versus each value at 7 days before administration. n = 5–12 for each group

mechanical allodynia appeared at 2 h after propentofylline administration and maintained for 24 h. Synergetic Analgesic Effect of EA and Propentofylline on Mechanical Allodynia A combined effect of EA and propentofylline on mechanical allodynia induced by TSS was examined. EA treatment was given 2 h after intrathecal administration of 0.1 or 1 lg propentofylline by lumbar puncture when the drug began exerting analgesic effect. The PWTs were measured at 1, 2 and 3 h after EA treatment. As shown in Fig. 2c and d, there was a synergetic effect of EA and the low dose propentofylline on mechanical allodynia induced by TSS. Intrathecal administration of propentofylline (0.1 lg, l.p.) without EA had no significant effect on mechanical allodynia induced by TSS (Fig. 2b). However, co-treatment of 0.1 lg propentofylline and EA elongated the duration of EA analgesia. Compared with the rats with 0.1 lg propentofylline alone, the value of MPE increased significantly in co-treatment rats at 3 h after EA treatment

(F1, 47 = 5.567, P \ 0.05; Fig. 2c). Further, the co-application of 1 lg propentofylline (l.p.) and EA produced more potent inhibition of allodynia than 1 lg propentofylline or EA treatment alone, especially at 2 h after EA treatment (F1, 44 = 5.151, P \ 0.05; Fig. 2d). To evaluate the combined analgesic effect of EA and propentofylline, we also calculated the analgesic ratio (%) in each group. The EA was considered to be valid only when MPE was more than 20%. In the EA group (n = 10), the analgesic ratio was 60%. However, when EA was combined with 0.1 lg (n = 12) or 1 lg (n = 6) propentofylline, the ratio was increased to 91.7 and 100%, respectively. In addition to the intrathecal administration of propentofylline, we further observed the effect of intraperitoneal administration (i.p.) of propentofylline (Fig. 2e, f). Propentofylline (1 mg/kg, i.p.) was given 2 h before EA treatment. Both the administration of propentofylline alone and the combined treatment of propentofylline with EA attenuated TSS-induced mechanical allodynia. However, the combined analgesic effect of propentofylline with EA was more significant (P \ 0.05, vs. pre-administration in

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PPF alone group; P \ 0.01, vs. pre-administration in combined group; n = 7 each group; Fig. 2e). The analgesic efficacy of propentofylline increased 22.2% at 1 h after EA treatment (Fig. 2f). Therefore, these results indicated that both the analgesic intensity and duration were strengthened when EA was combined with low dosage of propentofylline (l.p. or i.p.), suggesting a synergetically inhibitory effect of EA and propentofylline on mechanical allodynia induced by TSS. Inhibitory Effect of Propentofylline on Spinal Glial Activation By means of immunohistochemistry, we used Iba-1 and GFAP to label microglia and astrocytes in the spinal cord, respectively. TSS induced activation of microglia and astrocytes in the spinal cord as previously reported in other neuropathic pain models [14]. Expression of Iba-1 and GFAP were examined at 24 h after intrathecal propentofylline by lumbar puncture in TSS-operated rats. As shown in Fig. 3, Expression of Iba-1 and GFAP were obviously up-regulated in the spinal dorsal horn on 8 days after TSS. Increase of Iba-1- and GFAP-immunoreactivity by TSS was restored by propentofylline at the dosage of 1 and 10 lg, but not 0.1 lg. These results indicated that propentofylline inhibited activation of microglia and astrocytes induced by TSS.

Fig. 3 Effect of propentofylline on tetanic stimulation-induced increase in Iba-1immunoreactivity (Iba-1-IR) and GFAP-immunoreactivity (GFAP-IR) in the spinal dorsal horn. Photomicrographs showing Iba-1-IR (a) and GFAP-IR (b) in the ipsilateral spinal dorsal horn, respectively. Quantification of density of Iba-1-IR (c) and GFAP-IR (d) showing that single PPF (1 or 10 lg) suppressed the increase in Iba-1-IR and GFAPIR induced by tetanic stimulation. Measurements are expressed as mean ± SEM. ** P \ 0.01, *** P \ 0.001, versus the sham group; # P \ 0.05, ## P \ 0.01, ### P \ 0.001, 1 lg PPF or 10 lg PPF group versus the TSS group, one-way ANOVA followed by Holm-Sidak test. n = 3–4 for each group. Scale bars = 100 lm (a, b)

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Discussion Compelling evidence has demonstrated that tetanic stimulation of the sciatic nerve induces long-term potentiation in the spinal nociceptive neurons and persistent pain behaviors in rats [3, 21, 22]. Our recent study showed that the sciatic nerve was injured by the identical tetanic stimulation [21]. Therefore, tetanic stimulation-induced persistent pain characterizes as neuropathic pain. In present study, we demonstrated the synergetic analgesic action of propentofylline and EA on neuropathic pain induced by TSS. Considerable studies revealed that spinal glia is involved in the modulation of chronic pain [9, 10, 23] and EA analgesia [16, 17]. Therefore, by means of targeting glial function, therapeutic strategies to control chronic pain have been paid great attention. Some compounds such as fluorocitrate can disrupt glial function and decrease activation of microglia and/or astrocytes resulting in relieving pain responses in different chronic pain models [3, 17]. Unfortunately, most of them are not appropriate for human therapy due to side-effects. However, propentofylline, an inhibitor of glial activation, has been used to availably treat some human diseases [18, 19] and is capable of attenuating pain behavioral responses in a rodent model of neuropathic pain [14]. To probe into the potential value for clinical practice, we investigated the effect of propentofylline on EA analgesia. The present result showed that sub-efficient

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dose of propentofylline alone did not alter PWTs to mechanical stimulation, whereas a combination of propentofylline at sub-efficient dose and EA dramatically suppressed mechanical allodynia, suggesting a synergetic analgesia between EA and propentofylline via disturbing activity of microglia and astrocytes in the spinal cord. Such combination of low dose propentofylline and EA may provide a novel therapeutic method for improving the potency of EA analgesia. The mechanism underlying the synergetic analgesic interaction between EA and propentofylline is still unclear. Propentofylline can inhibit glial activation by strengthening of cyclic adenosine monophosphate (cAMP) signaling as a selective phosphodiesterase inhibitor and may directly regulate proinflammatory cytokine synthesis and release, such as interleukin-1 beta (IL-1b), tumor necrosis factor (TNF) [14]. Meanwhile, EA treatment intermittently can also inhibit activation of glial cells and decreases the levels of proinflammatory cytokine such as IL-1b [24]. Compelling evidence has demonstrated that opioid peptides and their l-, d- and j- receptors play a pivotal role in EA analgesia [4]. Given that glial cells express these three kinds of opioid receptors [25, 26], EA might affect glial cells directly or indirectly via opioid receptors. Therefore, it is conceivable that the synergetic analgesic interaction between propentofylline and EA may stem from blockade of activation of spinal glia and reducing release of painrelated substances in the spinal dorsal horn. In addition, propentofylline functions as an adenosine reuptake inhibitor [27]. Several reports showed that adenosine was implicated in the modulation of neuropathic pain. Intrathecal injection of adenosine 2A receptor agonist reversed neuropathic pain [28]. Propentofylline may activate adenosine and adenosine receptor and then augment inhibition of proinflammatory cytokine production [29]. Adenosine and adenosine receptor may also involve in the EA analgesia. EA-induced inhibition of spinal nociceptive neurons could be blocked by adenosine receptor antagonists [30]. Meanwhile, adenosine agonist could enhance opioid-mediated analgesia [31] and opioid peptides and their receptors play a pivotal role in EA analgesia. Taken together, adenosine system may also contribute to the synergetic analgesia between propentofylline and EA.

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18. Acknowledgments This work was supported by grants from National Basic Research Program of China (No. 2007CB512502 and 2007CB512303) and grants from National Nature Science Found of China (No. 30600178 and 30830044).

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