Transcutaneous Electrical Nerve Stimulation on Yongquan Acupoint Reduces CFA-Induced Thermal Hyperalgesia of Rats via Down-Regulation of ERK2 Phosphorylation and c-Fos Expression.код для вставкиСкачать
THE ANATOMICAL RECORD 293:1207–1213 (2010) Transcutaneous Electrical Nerve Stimulation on Yongquan Acupoint Reduces CFA-Induced Thermal Hyperalgesia of Rats via Down-Regulation of ERK2 Phosphorylation and c-Fos Expression LIN YANG, LIANXUE YANG, AND XIULAI GAO* Department of Anatomy, Histology and Embryology, Capital Medical University, Beijing, People’s Republic of China ABSTRACT Activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and its involvement in regulating gene expression in spinal dorsal horn, cortical and subcortical neurons by peripheral noxious stimulation contribute to pain hypersensitivity. Transcutaneous electrical nerve stimulation (TENS) is a treatment used in physiotherapy practice to promote analgesia in acute and chronic inﬂammatory conditions. In this study, a total number of 114 rats were used for three experiments. Effects of complete Freund’s adjuvant (CFA)-induced inﬂammatory pain hypersensitivity and TENS analgesia on ERK1/2 phosphorylation and c-Fos protein expression were examined by using behavioral test, Western blot, and immunostaining methods. We found that CFA injection caused an area of localized swelling, erythema, hypersensitivity to thermal stimuli, the decreased response time of hind paw licking (HPL), as well as upregulation of c-Fos protein expression and ERK2 phosphorylation in the ipsilateral spinal dorsal horn and the contralateral primary somatosensory area of cortex and the amygdala of rats. TENS on Yongquan acupoint for 20 min produced obvious analgesic effects as demonstrated with increased HPL to thermal stimuli of CFA-treated rats. In addition, TENS application suppressed the CFA-induced ERK2 activation and c-Fos protein expression. These results suggest that down-regulation of ERK2 phosphorylation and c-Fos expression were involved in TENS inhibition on CFA-induced therC 2010 Wileymal hyperalgesia of rats. Anat Rec, 293:1207–1213, 2010. V Liss, Inc. Key words: CFA-induced inﬂammatory pain; TENS application; ERK1/2; c-Fos Grant sponsor: National Natural Science Foundation of China; Grant number: 90209008; Grant sponsor: Beijing Natural Science Foundation; Grant number: 5072008. *Correspondence to: Xiulai Gao, MD, Department of Anatomy, Histology and Embryology, Capital Medical University, Beijing 100069, China E-mail: email@example.com C 2010 WILEY-LISS, INC. V Received 10 October 2009; Accepted 31 January 2010 DOI 10.1002/ar.21157 Published online 13 April 2010 in Wiley InterScience (www. interscience.wiley.com). 1208 YANG ET AL. Inﬂammatory and neuropathic pains are initiated by tissue damage/inﬂammation and nervous system lesions, and both are characterized by hypersensitivity at the site of damage and in adjacent normal tissue (Woolf and Salter, 2000). Up to now, intraplantar or joint injection of the complete Freund’s adjuvant (CFA) as an inﬂammatory pain model has been widely used (Kim et al., 2006). The nociceptive system in the central nervous system (CNS) includes the spinal dorsal horn, the primary somatosensory cortex (SI), and the amygdala (Woolf, 2007). It has been reported that both inﬂammation and nerve injury induce transcriptional changes in dorsal horn neurons, which are mediated by activation of the mitogen-activated protein kinases (MAPK)-cAMP response element binding protein cascade (McCarson and Krause, 1994; Hay et al., 1997). SI cortical neurons encode the intensity of tactile and nociceptive stimuli. Neurons in the medial temporal lobe areas, including the amygdala, are involved in learning the association between aversive and neutral stimuli in normal conditions (Buchel and Dolan, 2000; Petrovic et al., 2000; Petrovic et al., 2002). The amygdala may contribute to pain processing both directly by regulating nociceptive modulating systems in the brainstem and indirectly by controlling behavioral and autonomic output during pain (Petrovic et al., 2000; Petrovic et al., 2004). c-Fos is the most extensively investigated member of the immediate early gene family (IEGs). It has been considered a transcription factor and a cellular marker of neural activity to identify activated neurons response to peripheral noxious stimulation in the CNS. Numerous studies have demonstrated that noxious stimuli induce the expression of c-Fos in the SI area, the amygdala, and the spinal dorsal horn (Kwon et al., 2004; Roh et al., 2006). The activation of ERK1/2, a well known member of MAPK family, is mediated via the conserved Ras/Raf/MAPK pathway (Ji et al., 1999). Phosphorylated ERK1/2 (p-ERK1/2) has been also used as a marker of neural activation (Ji et al., 2002; Kawasaki et al., 2004). However, few studies have been carried out to investigate ERK1/2 activation and expression in the SI area and the amygdala after inﬂammatory pain stimulation. Transcutaneous electrical nerve stimulation (TENS) is a treatment that has been shown to be effective for pain relief in a variety of conditions (Bjordal et al., 2003; Chao et al., 2007). Acupuncture is also an important therapeutic strategy in traditional Chinese medicine (Zhang et al., 2003; Kim et al., 2005). However, acupuncture typically involves penetration of the skin on the speciﬁc points (named as acupoints) by a needle, and the analgesic effect varies in the different conditions. TENS on acupoints may serve as a relatively safe and noninvasive method to obtain the analgesic effect. In this study, we observed the TENS effects on phosphorylation and protein expression levels of ERK1/2 and c-Fos expression in the spinal dorsal horn, the SI area, and the amygdala of rats following CFA injection using TENS on Yongquan acupoint (KI 1). KI 1 is located on the sole of the foot, at the indentation near the front part, between the second and third metatarsal bones, one-third of the distance from the webs of the toes to the heel. KI 1 is usually applied to reduce pain on top of the head, blurry vision, throat numbness, and so forth (Lu and Liu, 1991). TABLE 1. Experimental groups 1 2 3 4 5 6 Saline only Saline þ Sham TENS Saline þ TENS CFA only CFA þ Sham TENS CFA þ TENS HPL test IHC test WB test 6 6 6 6 6 6 6 0 6 6 0 6 6 0 6 30a 0 12b Rats in different groups were sacriﬁced at 1 hr after saline or CFA injection. a Rats were sacriﬁced at ﬁve different time points (0.5, 1, 2, 24, and 48 hr after CFA injection). b Rats were sacriﬁced at two different time points (1 and 24 hr after CFA injection). TENS was applied for 20 min 0.5 and 23.5 hr after CFA injection. MATERIALS AND METHODS Animals and Drugs All experiments were carried out on speciﬁc pathogenfree adult (range, 12–16 weeks of age) male Wistar rats (weighing range, 180–230 g). The animal protocols were approved by University Institutional Animal Care and Use Committee of Capital Medical University, and were consistent with the NIH policy on the use of experimental animal (NIH Publications No. 80-23). A total of 114 rats were used in six different groups (see Table 1). Group 1 (Saline only): Eighteen rats were injected with 0.1 mL saline solution into the right ankle joint and tested or sacriﬁced 1 hr after injection. Out of 18 rats in Group 1, six rats were used for hind paw licking (HPL) test, six for immunohistochemistry (IHC), and six for western blot (WB), respectively; Group 2 (Saline þ sham TENS): Six rats were injected with 0.1 mL saline solution, treated with sham TENS for 20 min 0.5 hr after injection and used for HPL test at 1 hr after injection; Group 3 (Saline þ TENS): 18 rats were injected with 0.1 mL saline solution, applied TENS for 20 min 0.5 hr after injection and tested or sacriﬁced at 1 hr after injection. Out of 18 rats in Group 3, six rats were used for HPL test, six for IHC, and six for WB, respectively; Group 4 (CFA only): 12 rats were used for HPL test and IHC respectively, which were injected with 0.1 mL CFA and tested or sacriﬁced 1 hr after injection. Another 30 rats were used for WB, which were sacriﬁced at ﬁve different time points (0.5, 1, 2, 24, and 48 hr after CFA injection); Group 5 (CFA þ sham TENS): Six rats were injected with 0.1 mL CFA, treated with sham TENS and used for HPL test at 1 hr after injection; Group 6 (CFA þ TENS): 18 rats were injected with 0.1 mL CFA, applied TENS for 20 min 0.5 hr after injection and tested or sacriﬁced at 1 hr after injection. Another six rats were treated with TENS 23.5 hr after CFA injection and sacriﬁced 24 hr after CFA injection. CFA (0.1mL per animal, Gibco, USA) was injected into the right ankle joint to produce a pathological pain model as previously reported (Abbadie et al., 1994). TENS Application A TENS device (HANS LH202H, Beijing Huawei Company, Beijing, China) was used to induce antihyperalgesia. The following parameters were used: low (LF, 2 Hz) and high (HF, 100 Hz) frequency alternately, with 1209 TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION TABLE 2. Changes in response time of hind paw licking of rats after CFA injection and TENS application (N 5 6 per group) Control CFA injection Number (N) Before TENS stimulation (S) After sham TENS stimulation (S) After TENS stimulation (S) 18 18 8.65 1.68 5.12 1.29* 9.04 2.25 6.26 2.17* 14.72 3.84# 9.86 1.58*,# Versus control group, *P < 0.05; versus sham TENS stimulation, #P < 0.05; S, seconds. current intensity at range, 1–3 mA, and pulse duration at range, 0.2–0.6 ms. TENS was applied on KI 1 acupoint in the right hind limbs of rats at 0.5 hr or 23.5 hr after the injection of CFA or physiological saline solution and maintained for 20 min. Then the rats were tested for the response time of HPL or were sacriﬁced at the time point of 1 hr or 24 hr after CFA-injection. The parameters of TENS regarding frequency, pulse duration, application time, and intensity were in accordance to those used in routine physiotherapy practice (Han, 2003). Rats with sham TENS treatment were placed in a TENS device without electric current. Hyperalgesia was examined with measuring the response time of HPL to thermal stimuli using a hot plate analgesia meter (Hargreaves et al., 1988). The hot plate meter (YLS-6B, Huaibei Zhenghua Instruments & Equipments Company, China) was maintained at 52 0.2 C. Because of individual variations, the animals used in this study were chosen with the HPL response time shorter than 20 sec but longer than 6 sec under the normal condition. Immunostaining Study Rats were deeply anesthetized with chloral hydrate (300 mg/kg, ip), transcardially perfused with 0.1 M phosphate-buffered saline (PBS) followed by cold 4% paraformaldehyde in PBS. The lumbar-sacral enlargement of the spinal cord and the brain were postﬁxed in 4% paraformaldehyde and dehydrated in 20% sucrose solution overnight. Sections at 30 lm thickness were prepared for c-Fos IHC staining. Sections were incubated with 0.3% H2O2 in PBS (0.5 hr), blocked in 5% normal goat serum (60 min), and incubated overnight with primary antibody c-Fos (1:100, Santa Cruz), then with biotinylated goat anti-rabbit IgG. Finally, sections were developed with DAB (Sigma, USA) staining. c-Fos positive neurons in the spinal dorsal horn, the S1 area of the cortex, and the amygdala were counted in ﬁve representative sections. Five areas (126.50 lm2 per area) were chosen for cell counts from each section. Western Blotting Rats were anesthetized with chloral hydrate (250 mg/ kg, ip) and decapitated at each time point. The tissues from the bilateral spinal cord, the SI area of the cortex, and the amygdala were immediately removed. The sectioned tissues were homogenized at 4 C in homogenizing buffer and sonicated to dissolve the tissue completely as previously reported (Jiang et al., 2009). Then, the amounts of protein were determined by BCA kit (Pierce, USA). The levels of ERK1/2 phosphorylation (p-ERK1/2) and protein expression were determined using WB as described previously (Zhang et al., 2007). Samples containing an equal amount of total protein were loaded on 10% SDS-PAGE gel. After transferring to nitrocellulose membranes (Schleicher & Schuell, USA), unspeciﬁc binding was blocked by incubating in blocking buffer for 1 hr. The blots were then incubated with primary antibodies overnight at 4 C. The following antibodies at a 1:1,000 dilution were used: rabbit anti-mouse p-ERK1/2, rabbit anti-mouse total ERK1/2 (T-ERK1/2, Promega, USA), or mouse anti-b-actin (Sigma, USA). After incubating with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, protein was visualized using ECL-plus Kit (PerkinElmer Life Science, USA). Data Analysis Data were presented as mean SD (standard deviation). Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by individual post hoc multiple comparisons. A statistical difference was accepted as signiﬁcant at P < 0.05. RESULTS Effects of CFA Injection and TENS Application on Thermal Hyperalgesia of Rats CFA injection produced an area of localized swelling, erythema, and hypersensitivity to thermal stimuli, which persisted for the duration of experiment (48 hr). As shown in Table 2, the response time of HPL to thermal stimuli of CFA-injected rats at 1 hr decreased significantly (P < 0.05, N ¼ 6) when compared with that of control group. However, TENS application could increase the response time of HPL both in control and CFA injection groups signiﬁcantly (#P < 0.05, N ¼ 6). Effects of CFA Injection and TENS Application on c-Fos Expression in the Spinal Dorsal Horn, the SI Area of Cortex, and the Amygdala of Rats As shown in Fig. 1A, c-Fos protein expressed in the ipsilateral laminae I and II of the spinal dorsal horn, the contralateral SI area of cortex, and the amygdala. The numbers of c-Fos positive neurons in the ipsilateral spinal dorsal horn, the contralateral SI area of cortex, and the amygdala increased signiﬁcantly at 1 hr post CFA injection (P < 0.05, N ¼ 6, Fig. 1B and E). In addition, 20 min TENS stimulation also increased c-Fos protein expression signiﬁcantly in the ipsilateral spinal dorsal horn, the contralateral SI, and the 1210 YANG ET AL. Fig. 1. Effects of CFA injection and TENS application on c-Fos expressions in ipsilateral spinal dorsal horn, contralateral SI area of cortex, and amygdala of rats. Typical results of c-Fos immunostaining in the ipsilateral laminae I and II of the spinal dorsal horn, contralateral SI area of cortex, and amygdala of rats following injections of saline solution and sham transcutaneous electrical nerve stimulation (TENS, A), complete Freund’s adjuvant (CFA, B), TENS application (C), and TENS at 0.5 hr post CFA (D). *P < 0.05 versus control group, #P < 0.05 versus CFA-treated group, N ¼ 6 per group. amygdala (Fig. 1C and E). However, 20 min TENS stimulation started at 0.5 hr post CFA injection could reduce the number of c-Fos positive neurons in the ipsilateral spinal dorsal horn, but not in the contralateral SI area of cortex and the amygdala, when compared with that of CFA group at 1 hr post CFA injection (Fig. 1D and E). Effects of CFA Injection and TENS Application on ERK1/2 Phosphorylation and Protein Expression Levels in the Spinal Dorsal Horn, the SI Area of Cortex, and the Amygdala of Rats The representative immunoblotting results of p-ERK1/ 2 and T-ERK1/2 were shown in Fig. 2A (ipsilateral TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION 1211 Fig. 2. Typical Western blot results showed the changes in ERK1/2 phosphorylation and protein expression in ipsilateral spinal dorsal horn (A), contralateral SI area of cortex (B), and amygdala (C) of rats following CFA and TENS. ERK1/2 and b-actin were detected at 42/44 and 43 kDa, respectively. Control, saline solution, and sham TENS; P-0.5 hr, 0.5 hr post CFA; P-1 hr, 1 hr post CFA; P-2 hr, 2 hr post CFA; P-24 hr, 24 hr post CFA; P-48 hr, 48 hr post CFA; P-0.5 hr þ TENS, 20 min TENS at 0.5 hr post CFA; P-24 hr þ TENS, 20 min TENS at 24 hr post CFA. spinal dorsal horn), B (contralateral SI area of cortex), and C (contralateral amygdala). The bands of p-ERK1/2, T-ERK1/2 as well as b-actin could be detected at 44 kDa for ERK1, 42 kDa for ERK2, and 43 kDa for b-actin, respectively. As shown in Fig. 3A, the phosphorylation levels of ERK2, not ERK1, at 0.5 hr (118.9 5.2), 1 hr (115.2 5.4), 2 hr (128.6 8.4), 24 hr (123.1 4.6), and 48 hr (125.1 3.6) post CFA injection in ipsilateral spinal dorsal horn increased signiﬁcantly (P < 0.05) when compared with that of control group (100%, N ¼ 6 per group). However, TENS application could inhibit the increase of ERK2 phosphorylation in the ipsilateral spinal dorsal horn at 0.5 hr (105.7 4.6 vs. P-30 min, P < 0.05) and 24 hr (108.5 4.2 vs. P-24 h, P < 0.05) post CFA injection. Similarly, there were signiﬁcant increase of ERK2 phosphorylation both in contralateral SI (Fig. 2B and 3B) and amygdala (Fig. 2C and 3C) at 0.5 hr, 1 hr, 2 hr, 24 hr, and 48 hr post CFA injection. TENS application abolished the increase of ERK2 phosphorylation of CFAinjected rats (Fig. 2B, 2C, 3B and 3C). In addition, no changes of total ERK1/2 protein expressions were detected in the ipsilateral spinal dorsal horn, the contralateral SI area, and the amygdala of rats following CFA injection (data not shown). Fig. 3. Quantitative analysis of ERK1/2 phosphorylation level in ipsilateral spinal dorsal horn (A), and contralateral SI area of cortex (B), and amygdala (C) of rats following CFA and TENS. Fifty-four rats were randomly divided into the following nine groups: control (N ¼ 6), P-0.5 hr (N ¼ 6), P-1 hr (N ¼ 6), P-2 hr (N ¼ 6), P-24 hr (N ¼ 6), P-48 hr (N ¼ 6), P-0.5 hr þ TENS (N ¼ 6) and P-24 hr þ TENS (N ¼ 6). The results showed that TENS application suppressed CFA-induced ERK2 activation in the ipsilateral spinal cord, contralateral SI area of cortex, and amygdala of rats. *P < 0.05 versus control. DISCUSSION In this study, we undertook a detailed analysis on cFos protein expression and ERK1/2 phosphorylation levels in the spinal dorsal horn, the SI area of cortex, and the amygdala of rats in response to CFA injection and TENS stimulation. Our results demonstrated that CFA injection in ankle of rat in vivo causes signiﬁcant c-Fos expressions as well as ERK2 activation. The application of TENS stimulation on KI 1 could block the increases of c-Fos expression and ERK2 phosphorylation as well as an obvious analgesia, which could be detected via the increased response time of HPL in rats under thermal stimuli. Ji et al. (Ji et al., 1999; 2002) found that CFA injection of hind paw produced a persistent inﬂammation and a 1212 YANG ET AL. sustained ERK activation in neurons of the superﬁcial dorsal horn. Zhuang et al. (Zhuang et al., 2005) reported that ﬁfth lumbar spinal nerve ligation induced an immediate (<10 min) but transient (<6 hr) induction of ERK phosphorylation restricted to neurons in the superﬁcial dorsal horn. These ﬁndings suggested that the involvement of p-ERK in peripheral inﬂammatory pain hypersensitivity may be contributed to the regulation of target genes such as IEGs. In this study, we found that CFA induced a persistent ERK2 activation (>48 hr) not only in the spinal cord but also in the SI area of cortex and the amygdala. Our data also provided evidence that CFA injection causes an obvious increased expression of c-Fos in the spinal cord, the SI area of cortex, and the amygdala of rats. c-Fos is a very important IEGs member, which has been shown to take part in injury-related cellular mechanisms in many different systems including neuronal transport blockade (Guo et al., 2004; Wang et al., 2006). c-Fos induction by CFA had already been demonstrated in neurons of the spinal cord and cortex (Abbadie et al., 1994; Bellavance and Beitz, 1996; Cruz et al., 2007). Furthermore, we found that CFA injection induced c-Fos expressions in the amygdala. Amygdala is a complex subcortex structure, which mediates a speciﬁc aspect of emotional behaviors including the process of pain encoding and modulation. Studies have demonstrated that the amygdala has abundant opiates receptors and participates in both opioid analgesia and acupuncture analgesia (Fields, 2000; Napadow et al., 2007). The increases of c-Fos expression and ERK2 phosphorylation in the amygdala suggested that it may integrate the nociceptive information from the spinal cord and other cortical areas and then generate the emotional and behavioral responses on pain. Another important ﬁnding of this study is that TENS application on KI 1 could inhibit CFA-induced ERK2 activation and c-Fos expression. c-Fos and ERK expressions were reduced by ERK inhibitor in CFA-treated rats implicated the important role for ERK/c-Fos-dependent transduction pathways in acute and chronic modulation of nociceptive stimulation (Giles et al., 2007). In addition, TENS on acupoint induced an obvious analgesic effect by the increased HPL of CFAtreated rats. TENS has been used to promote analgesia for nearly 40 years. However, the use of TENS combined with acupoint has not been widespread despite of its prominent improvement in function of the affected region especially at pain relieving. Studies supported the hypothesis that therapeutic acupuncture was mediated by opioidergic and/or monoamingergic neurotransmission involving the brain stem, the thalamus, and the amygdala action (Garrison and Foreman, 1994; Dhond et al., 2007). Afferent spinal gating and the diffuse noxious inhibitory control may be involved in short-term analgesic effects of TENS stimulation (Carlsson, 2002). Our primary fMRI study demonstrated that the brain network associated with the amygdala implicated in both pain sensation and pain modulation, and acupuncture might change this amygdala-speciﬁc brain network into a functional state to affect pain perception and pain modulation (Qin et al., 2008). In conclusion, these ﬁndings suggest that down-regulation of ERK2 phosphorylation and c-Fos expression in the spinal dorsal horn as well as in the SI area of cortex and the amygdala were involved in TENS inhibition on CFA-induced thermal hyperalgesia of rats. 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