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Electroacupuncture suppresses spinal expression of neurokinin-1 receptors induced by persistent inflammation in rats
Neuroscience Letters 384 (2005) 339–343
Electroacupuncture suppresses spinal expression of neurokinin-1 receptors induced by persistent inflammation in rats Rui-Xin Zhang a,∗ , Bing Liu a , Jian-Tian Qiao b , Linbo Wang a , Ke Ren c , Brian M. Berman a , Lixing Lao a a
c
Center for Integrative Medicine, School of Medicine, University of Maryland, 3rd Floor, James Kernan Hospital, 2200 Kernan Drive, Baltimore, MD 21207, USA b Department of Neurobiology, Shanxi Medical University, Taiyuan 030001, Shanxi, PR China Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA Received 29 March 2005; received in revised form 2 May 2005; accepted 3 May 2005
Abstract It has been demonstrated that electroacupuncture (EA) significantly suppresses behavioral hyperalgesia in a rat model of persistent inflammatory pain and that neurokinin-1 (NK-1)/substance P (SP) receptors play important roles in nociception and hyperalgesia at the spinal cord level. The present study investigated spinal NK-1 receptor involvement in EA-produced suppression of hyperalgesia in a rat model of persistent inflammatory pain. The results showed that hind paw inflammation induced a significant increase of NK-1 receptor expression in the spinal dorsal horn and that this effect was significantly suppressed by EA. This suggests that EA-induced suppression of hyperalgesia is involved, at least partly, in the suppression of the spinal NK-1 receptors induced by sustained peripheral nociceptive input. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neurokinin-1 receptor; Hyperalgesia; Complete Freund’s adjuvant; Inflammatory pain; Spinal cord; Acupuncture
It has been demonstrated that neurokinin-1 (NK-1)/substance P (SP) receptors, activated by their ligand, SP [5,17], play important roles in nociception and hyperalgesia at the spinal cord level. For example, there is a significant upregulation of NK-1 in spinal dorsal horn neurons during CFA-induced hind paw inflammation [1,9]. Additionally, specific NK-1 receptor antagonists block noxious stimulus- or SP-evoked dorsal horn cell excitation [22] and suppress nociception-related increases of NK-1 receptor gene expression in dorsal horn [16]. Moreover, the ablation of spinal NK-1 receptor-containing neurons by a selective cytotoxin, SP–saporin conjugate, results in a reduction of thermal hyperalgesia and mechanical allodynia [19]. Our previous studies showed that electroacupuncture (EA) significantly suppresses the hyperalgesia induced by subcutaneous injection of complete Freund’s adjuvant (CFA) in rats [13,14], but the underlying mechanisms of this acupunc∗
Corresponding author. Tel.: +1 410 706 1582; fax: +1 410 706 1583. E-mail address: [email protected] (R.-X. Zhang).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.001
ture-produced attenuation of hyperalgesia were largely unclear. The hypothesis guiding the present study was that EA modulates the expression of NK-1 receptors in the spinal dorsal horn induced by persistent pain and thus suppresses the concomitant hyperalgesia in a rat model of CFA-induced persistent inflammation. Male Sprague–Dawley rats weighing 280–350 g (Harlan) were kept under controlled environmental conditions (22 ± 0.5 ◦ C; relative humidity, 40–60%; 12-h alternate light–dark cycle, food and water ad libitum). Inflammatory hyperalgesia was induced by injecting 0.08 ml of CFA (Sigma, suspended in an 1:1 oil/saline emulsion, 0.5 mg/ml heat-killed Mycobacterium tuberculosis) subcutaneously (s.c.) into the plantar surface of one hind paw of the rat using a 25 gauge hypodermal needle [14]. The animals were gently handled for 30 min each day for 2–3 days to habituate them to touch before the experiments. The animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
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Rats were randomly divided into the following groups (n = 8/per group): (1) saline-injected plus sham EA; (2) CFAinjected plus sham EA; (3) saline-injected plus EA treatment; (4) CFA-injected plus EA treatment. The EA treatment was given as in our earlier studies [14]. On the day of the CFA injection, two 20-min EA treatments were given: a prophylactic treatment immediately after the injection and a second treatment 2 h later. Two more 20-min EA treatments were given again on day 2 post-CFA injection. The EA parameters were 10 Hz at 3 mA and 0.1 ms pulse width, which showed significant suppression of both hyperalgesia and c-Fos protein expression in our previous studies with the rat model of inflammatory pain [13,14,28]. The equivalent of human acupoint Huantiao, also known as the 30th acupoint on the Gallbladder Meridian (GB30), on the rat’s hind limb was treated on the side ipsilateral to the CFA injection. GB30 was chosen based on traditional Chinese medicine (TCM) meridian theory [20] and its successful use in previous studies [13,14,25]. To determine acupoint and EA parameter specificity (i.e. whether a specific acupoint and specific set of EA parameters produce the optimum therapeutic effect for a given condition), we tested and compared the effects of various acupoints and EA parameters on hindpaw inflammatory hyperalgesia in a prior study [14]. The data showed that EA (3 mA, 10 Hz, 0.1 ms, for 20 min) at acupoint GB30 produced significantly better anti-hyperalgesia than did EA at Waiguan (the fifth acupoint on the Triple Energizer Meridian, TE 5) or at sham points, one at the opposite aspect of GB30 and the other on the abdomen. In humans, GB30 is located at the junction of the lateral one-third and medial two-third of the distance between the greater trochanter and the hiatus of the sacrum; underneath are the sciatic nerve, inferior gluteal nerve, and gluteal muscles [4]. Comparable landmarks were used to locate GB30 in the rats. The transposition method for determining acupoints in animals has been shown to be effective [23]. Two disposable acupuncture needles (gauge #32, 0.5 in. with an interpolar distance of 4 mm) were swiftly inserted approximately 0.5 in. into GB30 on the inflamed side and connected to an electrical stimulator (A300 Pulsemaster, World Precision Instruments). While EA frequency was held constant, intensity was adjusted slowly over the period of approximately 2 min to the designated level of 2 mA. Mild muscle twitching was observed. During EA treatment, each rat was placed under an inverted clear plastic chamber (approximately 5 in. × 8 in. × 11 in.) but was neither restrained nor given any anesthetic. The animals remained awake and still and gave no observable signs of distress during the treatment [14]. For sham control, acupuncture needles were similarly inserted but without electrical stimulation or manual needle manipulation. This form of sham EA showed little antihyperalgesic effect in our previous study [14] and is used as a control for non-specific needling effects. Hind paw withdrawal latency (PWL) was tested once prior to the CFA injection and twice after EA treatments, on the
day of injection and on day 2 post-injection, using a method developed previously [8,13]. Mean PWL was established by averaging the latency of four tests with a 5-min interval between each test. The investigator who performed the behavioral tests was blind to the treatment assignments. NK-1 receptor expression experiments were conducted on day 3 after the CFA injection. Rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused transcardially with 100 ml of saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The lumbar (L4) spinal cord was removed, immersed in the same fixative for 2 h and transferred to a 30% sucrose in phosphate buffer for overnight cryoprotection. Thirty micrometer-thick sections were immunostained with anti-NK-1 receptor antibody (1:2000, Chemicon) using the standard ABC method [27]. Five sections were randomly selected from each animal for analysis. The stained sections were analyzed under a Nikon E600 microscope to quantify the optical density (NIH Image 1.6) of NK-1 receptor-like immunoreactive (LI) staining in the superficial laminae of the spinal cord. Spinal sections were photographed using a 4× objective lens. Threshold intensity was determined for each image and most background noise was suppressed. A rectangular box of constant size was placed over the entire width of the superficial dorsal horn, and the number of pixels and the mean grey value within the box were determined on both sides of the spinal cord. Density values of each section were calculated by multiplying pixels by mean grey value, averaged to yield a value for each animal, and mean and standard error were determined for the group. The experimenter responsible for image collection and quantification was blinded to the treatment groups. Data were presented as mean ± S.E. Behavioral data were analyzed with repeated measure analysis of variance (ANOVA). Immunohistochemical data were analyzed with ANOVA followed by the Dunnett’s post hoc test. P < 0.05 was considered significant. Before CFA treatment, mean baseline PWL to noxious heat stimuli was similar in the left hind paws of CFA plus sham EA and CFA plus EA rats (Fig. 1, P > 0.05), and there was no significant difference in baseline PWL between left and right paws. To simplify the illustration, the contralateral PWLs are omitted. After a 0.08 ml injection of CFA into the left hind paw, PWL of the injected paw was significantly shorter than baseline, while that of the contralateral hind paw remained at the pre-CFA level. Compared to sham EA, EA at GB30 significantly lengthened PWL of the CFA-injected hind paw at 2.5 h (6.51 ± 0.18 versus 5.35 ± 0.41, P < 0.05) and 2 days (8.26 ± 0.47 versus 6.90 ± 0.25, P < 0.05) postCFA, which shows an anti-hyperalgesic effect (Fig. 1). Since recovery from CFA-induced inflammation begins on day 1 post-injection, PWLs on day 2 was longer than that at 2.5 h. The two saline-injected groups showed no hyperalgesia following saline injection, and their PWLs showed no significant changes after EA or sham EA (data not shown).
R.-X. Zhang et al. / Neuroscience Letters 384 (2005) 339–343
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Fig. 1. Comparisons of hind paw withdrawal latencies (PWLs) between EA treatment (n = 8) and sham EA (n = 8) as measured prior to CFA injection (baseline) and 2.5 h and 2 days post-CFA injection. ∗ P < 0.05 in comparison to sham EA PWLs.
Fig. 3. Histograms of summarized data showing the effects of EA treatment on NK-1 receptor immunoreactivity in spinal superficial laminae following CFA or saline injection into left hind paws. ** P < 0.01 compared to the ipsilateral and the contralateral sides of all groups, N = 5 each group.
Immunohistochemical examination shows that neither saline injection plus sham EA nor saline injection plus EA affected NK-1 receptor immunoreactive profiles on either side of the spinal cord dorsal horn (Fig. 2A and B). However, CFA injection plus sham EA elicited a dramatic increase of NK1 receptor immunoreactivity in the superficial dorsal horn (laminae I and II) of the lumbar spinal cord ipsilateral to the
CFA injection (Fig. 2C and E) compared to that of the contralateral side and that of the saline-injected groups. Moreover, dense CFA-induced NK-1 receptor immunoreactivity was suppressed by EA (Fig. 2D). Fig. 3 summarizes the statistical data (n = 5). Please note the abrupt increase of NK1 receptor immunoreactivity in the superficial dorsal horn
Fig. 2. Representative photomicrographs showing the effect of EA, applied immediately after CFA injection and again 2 days after CFA injection, on the immunoreactivity of spinal NK-1 receptors in the spinal dorsal horn of rats with CFA-induced hind paw inflammation. Neither saline plus sham EA (A) nor saline plus EA (B) induced immunoreactivity changes in these receptors in the ipsilateral spinal superficial laminae compared to those on the contralateral side; EA treatment (D), but not sham EA (C), suppressed the CFA-enhanced immunoreactivity of these receptors in the ipsilateral side. (E) and (F) are high magnifications of ipsilateral superficial laminae of dorsal horn shown in (C) and (D). Scale bars are 100 m in (A)–(D), and 20 m in (E) and (F). Arrows point to superficial laminae.
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(laminae I and II) of the lumbar spinal cord following CFA injection (** P < 0.01) and the significant suppression of this neurochemical response after EA treatment. Our data show that EA significantly suppressed the thermal hyperalgesiaof CFA-induced persistent pain rat model, as we reported previously [13,14]. A previous study using female rats and the hot plate test demonstrated that EA did not show inhibitory effects on thermal hyperalgesia in the CFA model [10]. The discrepancy may be due to the sexual difference and/or to testing method. It is known that sexual hormones have significant effects on pain [12]. Besides, it should be noted that hyperalgesia was induced in both ipsilateral and contralateral hind paws when CFA at 500 g [18] or carrageenan at 3% (100 l) [21] was injected unilaterally, while it was induced only ipsilaterally when 1% carrageenan was injected [21]. In our study, 40 g of CFA was injected into one hind paw and only the injected paw showed hyperalgesia, which is consistent with our previous studies [14]. Therefore, the contralateral side was used as control in the present study. Our present study also demonstrates that the suppressive EA action was concomitantly paralleled by a significant inhibition of CFA-induced NK-1 up-expression in the superficial laminae of the dorsal horn ipsilateral to the CFAinjection. It has been reported that NK-1 receptors play important roles in the transmission of noxious inputs and the development of hyperalgesia at the spinal level [1,6,9,19] and that NK-1 receptor immunoreactive density parallels behavioral hyperalgesia [7]. Therefore, the present study strongly suggests that EA suppression of NK-1 receptor expression is one of the underlying mechanisms by which EA attenuates hyperalgesia induced by sustained nociceptive inputs. Endogenous opioids may be involved in EA suppression of NK-1 receptor expression at the spinal level. Our recent study shows that EA activates the endorphin/endomorphin and enkephalin systems that act, respectively, on mu- and delta-opioid receptors during persistent pain [28]. Furthermore, activation of the mu- and delta-opioid receptors blocks the release of SP [2] and inhibits the expression of mRNA encoding NK-1 receptors in the dorsal horn [15], which are up-regulated by SP [24]. Given that EA inhibits a tooth pulp stimulation-evoked increase in the release of immunoreactive SP [26], the present results suggest that EA-produced release of opioids may inhibit the release of SP, leading to the suppression of NK-1 receptor expression and thus the suppression of hyperalgesia. In addition, it has been reported that ketamine potentiates EA-antiallodynia [11]. EA also reverses the down-regulation of NMDAR1 mRNA induced by chronic constriction injury (CCI) to the sciatic nerve [3]. Therefore, we do not exclude the possibility that EA may exert its action through NMDA receptors. In conclusion, EA decreases persistent inflammationinduced NK-1 receptor expression, which may be one of the mechanisms underlying EA-produced anti-hyperalgesia.
Acknowledgements We would like to thank Dr. Lyn Lowry for her editorial support. This work was supported by DRIF Funding, School of Medicine, University of Maryland.
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Electroacupuncture suppresses spinal expression of neurokinin-1 receptors induced by persistent inflammation in rats Rui-Xin Zhang a,∗ , Bing Liu a , Jian-Tian Qiao b , Linbo Wang a , Ke Ren c , Brian M. Berman a , Lixing Lao a a
c
Center for Integrative Medicine, School of Medicine, University of Maryland, 3rd Floor, James Kernan Hospital, 2200 Kernan Drive, Baltimore, MD 21207, USA b Department of Neurobiology, Shanxi Medical University, Taiyuan 030001, Shanxi, PR China Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA Received 29 March 2005; received in revised form 2 May 2005; accepted 3 May 2005
Abstract It has been demonstrated that electroacupuncture (EA) significantly suppresses behavioral hyperalgesia in a rat model of persistent inflammatory pain and that neurokinin-1 (NK-1)/substance P (SP) receptors play important roles in nociception and hyperalgesia at the spinal cord level. The present study investigated spinal NK-1 receptor involvement in EA-produced suppression of hyperalgesia in a rat model of persistent inflammatory pain. The results showed that hind paw inflammation induced a significant increase of NK-1 receptor expression in the spinal dorsal horn and that this effect was significantly suppressed by EA. This suggests that EA-induced suppression of hyperalgesia is involved, at least partly, in the suppression of the spinal NK-1 receptors induced by sustained peripheral nociceptive input. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neurokinin-1 receptor; Hyperalgesia; Complete Freund’s adjuvant; Inflammatory pain; Spinal cord; Acupuncture
It has been demonstrated that neurokinin-1 (NK-1)/substance P (SP) receptors, activated by their ligand, SP [5,17], play important roles in nociception and hyperalgesia at the spinal cord level. For example, there is a significant upregulation of NK-1 in spinal dorsal horn neurons during CFA-induced hind paw inflammation [1,9]. Additionally, specific NK-1 receptor antagonists block noxious stimulus- or SP-evoked dorsal horn cell excitation [22] and suppress nociception-related increases of NK-1 receptor gene expression in dorsal horn [16]. Moreover, the ablation of spinal NK-1 receptor-containing neurons by a selective cytotoxin, SP–saporin conjugate, results in a reduction of thermal hyperalgesia and mechanical allodynia [19]. Our previous studies showed that electroacupuncture (EA) significantly suppresses the hyperalgesia induced by subcutaneous injection of complete Freund’s adjuvant (CFA) in rats [13,14], but the underlying mechanisms of this acupunc∗
Corresponding author. Tel.: +1 410 706 1582; fax: +1 410 706 1583. E-mail address: [email protected] (R.-X. Zhang).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.001
ture-produced attenuation of hyperalgesia were largely unclear. The hypothesis guiding the present study was that EA modulates the expression of NK-1 receptors in the spinal dorsal horn induced by persistent pain and thus suppresses the concomitant hyperalgesia in a rat model of CFA-induced persistent inflammation. Male Sprague–Dawley rats weighing 280–350 g (Harlan) were kept under controlled environmental conditions (22 ± 0.5 ◦ C; relative humidity, 40–60%; 12-h alternate light–dark cycle, food and water ad libitum). Inflammatory hyperalgesia was induced by injecting 0.08 ml of CFA (Sigma, suspended in an 1:1 oil/saline emulsion, 0.5 mg/ml heat-killed Mycobacterium tuberculosis) subcutaneously (s.c.) into the plantar surface of one hind paw of the rat using a 25 gauge hypodermal needle [14]. The animals were gently handled for 30 min each day for 2–3 days to habituate them to touch before the experiments. The animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
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Rats were randomly divided into the following groups (n = 8/per group): (1) saline-injected plus sham EA; (2) CFAinjected plus sham EA; (3) saline-injected plus EA treatment; (4) CFA-injected plus EA treatment. The EA treatment was given as in our earlier studies [14]. On the day of the CFA injection, two 20-min EA treatments were given: a prophylactic treatment immediately after the injection and a second treatment 2 h later. Two more 20-min EA treatments were given again on day 2 post-CFA injection. The EA parameters were 10 Hz at 3 mA and 0.1 ms pulse width, which showed significant suppression of both hyperalgesia and c-Fos protein expression in our previous studies with the rat model of inflammatory pain [13,14,28]. The equivalent of human acupoint Huantiao, also known as the 30th acupoint on the Gallbladder Meridian (GB30), on the rat’s hind limb was treated on the side ipsilateral to the CFA injection. GB30 was chosen based on traditional Chinese medicine (TCM) meridian theory [20] and its successful use in previous studies [13,14,25]. To determine acupoint and EA parameter specificity (i.e. whether a specific acupoint and specific set of EA parameters produce the optimum therapeutic effect for a given condition), we tested and compared the effects of various acupoints and EA parameters on hindpaw inflammatory hyperalgesia in a prior study [14]. The data showed that EA (3 mA, 10 Hz, 0.1 ms, for 20 min) at acupoint GB30 produced significantly better anti-hyperalgesia than did EA at Waiguan (the fifth acupoint on the Triple Energizer Meridian, TE 5) or at sham points, one at the opposite aspect of GB30 and the other on the abdomen. In humans, GB30 is located at the junction of the lateral one-third and medial two-third of the distance between the greater trochanter and the hiatus of the sacrum; underneath are the sciatic nerve, inferior gluteal nerve, and gluteal muscles [4]. Comparable landmarks were used to locate GB30 in the rats. The transposition method for determining acupoints in animals has been shown to be effective [23]. Two disposable acupuncture needles (gauge #32, 0.5 in. with an interpolar distance of 4 mm) were swiftly inserted approximately 0.5 in. into GB30 on the inflamed side and connected to an electrical stimulator (A300 Pulsemaster, World Precision Instruments). While EA frequency was held constant, intensity was adjusted slowly over the period of approximately 2 min to the designated level of 2 mA. Mild muscle twitching was observed. During EA treatment, each rat was placed under an inverted clear plastic chamber (approximately 5 in. × 8 in. × 11 in.) but was neither restrained nor given any anesthetic. The animals remained awake and still and gave no observable signs of distress during the treatment [14]. For sham control, acupuncture needles were similarly inserted but without electrical stimulation or manual needle manipulation. This form of sham EA showed little antihyperalgesic effect in our previous study [14] and is used as a control for non-specific needling effects. Hind paw withdrawal latency (PWL) was tested once prior to the CFA injection and twice after EA treatments, on the
day of injection and on day 2 post-injection, using a method developed previously [8,13]. Mean PWL was established by averaging the latency of four tests with a 5-min interval between each test. The investigator who performed the behavioral tests was blind to the treatment assignments. NK-1 receptor expression experiments were conducted on day 3 after the CFA injection. Rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused transcardially with 100 ml of saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The lumbar (L4) spinal cord was removed, immersed in the same fixative for 2 h and transferred to a 30% sucrose in phosphate buffer for overnight cryoprotection. Thirty micrometer-thick sections were immunostained with anti-NK-1 receptor antibody (1:2000, Chemicon) using the standard ABC method [27]. Five sections were randomly selected from each animal for analysis. The stained sections were analyzed under a Nikon E600 microscope to quantify the optical density (NIH Image 1.6) of NK-1 receptor-like immunoreactive (LI) staining in the superficial laminae of the spinal cord. Spinal sections were photographed using a 4× objective lens. Threshold intensity was determined for each image and most background noise was suppressed. A rectangular box of constant size was placed over the entire width of the superficial dorsal horn, and the number of pixels and the mean grey value within the box were determined on both sides of the spinal cord. Density values of each section were calculated by multiplying pixels by mean grey value, averaged to yield a value for each animal, and mean and standard error were determined for the group. The experimenter responsible for image collection and quantification was blinded to the treatment groups. Data were presented as mean ± S.E. Behavioral data were analyzed with repeated measure analysis of variance (ANOVA). Immunohistochemical data were analyzed with ANOVA followed by the Dunnett’s post hoc test. P < 0.05 was considered significant. Before CFA treatment, mean baseline PWL to noxious heat stimuli was similar in the left hind paws of CFA plus sham EA and CFA plus EA rats (Fig. 1, P > 0.05), and there was no significant difference in baseline PWL between left and right paws. To simplify the illustration, the contralateral PWLs are omitted. After a 0.08 ml injection of CFA into the left hind paw, PWL of the injected paw was significantly shorter than baseline, while that of the contralateral hind paw remained at the pre-CFA level. Compared to sham EA, EA at GB30 significantly lengthened PWL of the CFA-injected hind paw at 2.5 h (6.51 ± 0.18 versus 5.35 ± 0.41, P < 0.05) and 2 days (8.26 ± 0.47 versus 6.90 ± 0.25, P < 0.05) postCFA, which shows an anti-hyperalgesic effect (Fig. 1). Since recovery from CFA-induced inflammation begins on day 1 post-injection, PWLs on day 2 was longer than that at 2.5 h. The two saline-injected groups showed no hyperalgesia following saline injection, and their PWLs showed no significant changes after EA or sham EA (data not shown).
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Fig. 1. Comparisons of hind paw withdrawal latencies (PWLs) between EA treatment (n = 8) and sham EA (n = 8) as measured prior to CFA injection (baseline) and 2.5 h and 2 days post-CFA injection. ∗ P < 0.05 in comparison to sham EA PWLs.
Fig. 3. Histograms of summarized data showing the effects of EA treatment on NK-1 receptor immunoreactivity in spinal superficial laminae following CFA or saline injection into left hind paws. ** P < 0.01 compared to the ipsilateral and the contralateral sides of all groups, N = 5 each group.
Immunohistochemical examination shows that neither saline injection plus sham EA nor saline injection plus EA affected NK-1 receptor immunoreactive profiles on either side of the spinal cord dorsal horn (Fig. 2A and B). However, CFA injection plus sham EA elicited a dramatic increase of NK1 receptor immunoreactivity in the superficial dorsal horn (laminae I and II) of the lumbar spinal cord ipsilateral to the
CFA injection (Fig. 2C and E) compared to that of the contralateral side and that of the saline-injected groups. Moreover, dense CFA-induced NK-1 receptor immunoreactivity was suppressed by EA (Fig. 2D). Fig. 3 summarizes the statistical data (n = 5). Please note the abrupt increase of NK1 receptor immunoreactivity in the superficial dorsal horn
Fig. 2. Representative photomicrographs showing the effect of EA, applied immediately after CFA injection and again 2 days after CFA injection, on the immunoreactivity of spinal NK-1 receptors in the spinal dorsal horn of rats with CFA-induced hind paw inflammation. Neither saline plus sham EA (A) nor saline plus EA (B) induced immunoreactivity changes in these receptors in the ipsilateral spinal superficial laminae compared to those on the contralateral side; EA treatment (D), but not sham EA (C), suppressed the CFA-enhanced immunoreactivity of these receptors in the ipsilateral side. (E) and (F) are high magnifications of ipsilateral superficial laminae of dorsal horn shown in (C) and (D). Scale bars are 100 m in (A)–(D), and 20 m in (E) and (F). Arrows point to superficial laminae.
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(laminae I and II) of the lumbar spinal cord following CFA injection (** P < 0.01) and the significant suppression of this neurochemical response after EA treatment. Our data show that EA significantly suppressed the thermal hyperalgesiaof CFA-induced persistent pain rat model, as we reported previously [13,14]. A previous study using female rats and the hot plate test demonstrated that EA did not show inhibitory effects on thermal hyperalgesia in the CFA model [10]. The discrepancy may be due to the sexual difference and/or to testing method. It is known that sexual hormones have significant effects on pain [12]. Besides, it should be noted that hyperalgesia was induced in both ipsilateral and contralateral hind paws when CFA at 500 g [18] or carrageenan at 3% (100 l) [21] was injected unilaterally, while it was induced only ipsilaterally when 1% carrageenan was injected [21]. In our study, 40 g of CFA was injected into one hind paw and only the injected paw showed hyperalgesia, which is consistent with our previous studies [14]. Therefore, the contralateral side was used as control in the present study. Our present study also demonstrates that the suppressive EA action was concomitantly paralleled by a significant inhibition of CFA-induced NK-1 up-expression in the superficial laminae of the dorsal horn ipsilateral to the CFAinjection. It has been reported that NK-1 receptors play important roles in the transmission of noxious inputs and the development of hyperalgesia at the spinal level [1,6,9,19] and that NK-1 receptor immunoreactive density parallels behavioral hyperalgesia [7]. Therefore, the present study strongly suggests that EA suppression of NK-1 receptor expression is one of the underlying mechanisms by which EA attenuates hyperalgesia induced by sustained nociceptive inputs. Endogenous opioids may be involved in EA suppression of NK-1 receptor expression at the spinal level. Our recent study shows that EA activates the endorphin/endomorphin and enkephalin systems that act, respectively, on mu- and delta-opioid receptors during persistent pain [28]. Furthermore, activation of the mu- and delta-opioid receptors blocks the release of SP [2] and inhibits the expression of mRNA encoding NK-1 receptors in the dorsal horn [15], which are up-regulated by SP [24]. Given that EA inhibits a tooth pulp stimulation-evoked increase in the release of immunoreactive SP [26], the present results suggest that EA-produced release of opioids may inhibit the release of SP, leading to the suppression of NK-1 receptor expression and thus the suppression of hyperalgesia. In addition, it has been reported that ketamine potentiates EA-antiallodynia [11]. EA also reverses the down-regulation of NMDAR1 mRNA induced by chronic constriction injury (CCI) to the sciatic nerve [3]. Therefore, we do not exclude the possibility that EA may exert its action through NMDA receptors. In conclusion, EA decreases persistent inflammationinduced NK-1 receptor expression, which may be one of the mechanisms underlying EA-produced anti-hyperalgesia.
Acknowledgements We would like to thank Dr. Lyn Lowry for her editorial support. This work was supported by DRIF Funding, School of Medicine, University of Maryland.
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