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Acupuncture decreases nitric oxide synthase expression in periaqueductal gray area of rats with streptozotocin-induced diabetes
Neuroscience Letters 337 (2003) 155–158 www.elsevier.com/locate/neulet
Acupuncture decreases nitric oxide synthase expression in periaqueductal gray area of rats with streptozotocin-induced diabetes Mi-Hyeon Jang a, Min-Chul Shin a, Gyo-Sung Koo b, Choong-Yeol Lee b, Ee-Hwa Kim c, Chang-Ju Kim a,* a
Department of Physiology, College of Medicine, Kyung Hee University, #1 Hoigi-dong, Dongdaemoon-gu, Seoul 130-701, South Korea b Department of Physiology, College of Oriental Medicine, Kyungwon University, Sungnam, South Korea c Department of Meridianology, College of Oriental Medicine, Semyung University, Jechon, South Korea Received 4 October 2002; accepted 12 November 2002
Abstract Acupuncture has been used as a clinical treatment in Oriental medicine for various diseases including diabetes mellitus, one of the most common metabolic disorders in humans. In the present study, the effect of acupuncture on the expressions of neuronal nitric oxide synthase (nNOS) and nitric oxide synthase (NOS) in the dorsolateral periaqueductal gray (DL-PAG) area of rats with streptozotocin (STZ)-induced diabetes was investigated via nNOS immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. Enhanced expression of nNOS and NOS was detected in the DL-PAG of rats with STZ-induced diabetes, and acupunctural treatment at Zusanli acupoint suppressed the diabetes-induced enhancement in the expression of nNOS and NOS. The present results demonstrate that acupuncture is effective in the modulation of the expression of nNOS and NOS in the DL-PAG under diabetic conditions. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acupuncture; Diabetes; Nitric oxide synthase; Neuronal nitric oxide synthase; Dorsolateral periaqueductal gray
Acupuncture has been used as a clinical treatment for various diseases in Oriental medicine. Acupuncture is known to possess many effects such as promotion of homeostasis, improvement in brain circulation, and pain control [8,17]. In addition, it has been reported that electroacupunctural stimulation is effective in treating diabetes [2,9]. Diabetes mellitus is one of the most common metabolic disorders in humans. In addition to the diabetic condition itself, numerous secondary complications are associated with the illness. It has been established that streptozotocin (STZ)-induced diabetes in rats produces a condition similar to the clinical form of diabetes mellitus, with severe peripheral neuropathy and inflammation [5]. Courteix et al. [3] reported that induction of insulin-dependent diabetes mellitus in rats causes allodynia and hyperalgesia. Nitric oxide (NO), synthesized from l-arginine through calcium-dependent pathways by nitric oxide synthase * Corresponding author. Tel.: 182-2-961-0407; fax: 182-2-9642195. E-mail address: [email protected] (C.-J. Kim).
(NOS), is a free radical with signaling functions in the central nervous system (CNS). NO has been implicated in the regulation of autonomic functions, and it has been shown to play important roles in the neural, vascular, and immune systems [4]. Several isoforms of NOS exist and fall into three major classes: inducible NOS, endothelial NOS, and neuronal NOS (nNOS). Of these, nNOS is mainly expressed in the CNS and has been implicated in signal transmission [4,7]. The midbrain periaqueductal gray (PAG) is believed to be an important component in the endogenous pain control system [1]. In immunohistochemical studies, the presence of NOS-staining neurons has been demonstrated in the dorsolateral PAG (DL-PAG). In the present study, the effect of acupuncture at the Zusanli acupoint (ST36) on the expression of nNOS and NOS in the DL-PAG was investigated via nNOS immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, which takes advantage of the fact that NADPH-d-positive neurons are the same as those containing NOS [7].
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S03 04 -3 94 0( 02 ) 01 318 - 6
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Male Sprague–Dawley rats weighing 200 ^ 10 g (6 weeks of age) were used for the experiment. Each animal was housed at a controlled temperature (20 ^ 2 8C) and was maintained under light–dark cycles, each cycle consisting of 12 h of light and 12 h of darkness (lights on from 07:00 h to 19:00 h), with food and water made available ad libitum. The experimental procedures were performed in accordance with the animal care guidelines of the NIH and the Korean Academy of Medical Sciences. Animals were divided into six groups: the control group, the nondiabetic and Zusanliacupunctured group, the nondiabetic and nonacupointacupunctured group, the STZ-induced-diabetes group, the STZ-induced-diabetes and Zusanli-acupunctured group, and the STZ-induced-diabetes and nonacupoint-acupunctured group (n ¼ 5 in each group). To induce diabetes in the experimental animals, a single intraperitoneal injection of STZ (50 mg/kg in saline; Sigma Chemical Co., St. Louis, MO) was given to each animal on the first day of the experiment, while animals of the control group and the nondiabetic groups received equivalent amounts of normal saline. Blood glucose levels were determined 2 days after STZ injection using a blood glucose analyzer (Arkray, Kyoto, Japan). Only the animals with blood glucose levels of 300 mg/dl or higher were used in this study. In the acupunctured groups, acupunctural treatment was given to each animal twice daily (10:00 a.m. and 5:00 p.m.) for 5 days, starting on the third day of the experiment. For acupunctural stimulation at Zusanli, stainless acupuncture needles of 0.3 mm diameter were bilaterally inserted into the locus of Zusanli, located 5 mm lateral and distal to the anterior tubercle of the tibia, and left in place for 20 min [9]. For treatment of animals of the nonacupoint-acupunctured groups, needles of the same diameter were inserted bilaterally at the hip and left in place for 20 min. All animals were sacrificed on the 7th day of the experiment. Animals were weighed and overdosed with Zoletil 50 w (10 mg/kg, i.p.; Vibac Laboratories, Carros, France). After a complete lack of response was observed, the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS) and then with 4% paraformaldehyde in 100 mM phosphate buffer (PB) at pH 7.4. The brains were dissected, postfixed in the same fixative overnight, and transferred into a 30% sucrose solution for cryoprotection. Serial coronal sections of 40 mm thickness were made using a freezing microtome (Leica, Nussloch, Germany). For analyzing the level of nNOS expression, ten sections on average were selected from each brain in the region spanning from Bregma 25.30– 2 8.30 mm according to the atlas by Paxinos and Watson [14]. Free-floating tissue sections were washed twice in 50 mM PBS and were then permeabilized in 0.2% Triton X-100 for 30 min. After washing twice with PBS, sections were incubated overnight with mouse anti-nNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:1000. Sections were washed twice in PBS and incubated for 1 h with biotinylated anti-rabbit antibody (1:200). Bound secondary antibody was
then amplified with Vector Elite ABC kit (Vector Laboratories, Burlingame, CA). The antibody-biotin-avidin-peroxidase complexes were visualized using 0.05% diaminobenzidine. The sections were mounted onto gelatinized glass slides and air-dried, and cover slides were mounted using Permount w. For NADPH-d activity, free-floating sections were incubated at 378C for 60 min in 100 mM PB containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 0.1 mg/ ml b-NADPH. The sections were then washed three times with PBS and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount w. The area of PAG was measured using Image-Pro wPlus image analyzer (Media Cybernetics Inc., Silver Spring, MD). The total numbers of nNOS-positive and NADPHd-positive neurons in the PAG were counted hemilaterally under a light microscope (Olympus, Tokyo, Japan), and the results were expressed as numbers of nNOS-positive and NADPH-d-positive cells per mm 2 of the area of the PAG region. Data was analyzed using SPSS by one-way analysis of variance followed by Scheffe´ ’s post-hoc test, and results were expressed as mean ^ standard error mean (SEM). Differences were considered significant for P , 0:05. In the present study, nNOS-positive and NADPH-d-positive cells were mainly localized in the dorsolateral area of the PAG, consistent with the findings of Rodella et al. [16]. The number of nNOS-positive cells in the animals of the control group (which received neither STZ nor acupunctural treatment) was 365.89 ^ 12.10/mm 2, and the figure for the nondiabetic and Zusanli-acupunctured group and the nondiabetic and nonacupoint-acupunctured group was 372.29 ^ 15.34/mm 2 and 342.11 ^ 4.43/ mm 2, respectively. This number was increased to 468.12 ^ 5.35/ mm 2 in the STZ-induced diabetes group (the animals of which did not receive any acupunctural treatment) but was decreased again to 370.43 ^ 6.08/mm 2 in the STZinduced-diabetes and Zusanli-acupunctured group; the number was 443.00 ^ 12.37/mm 2 in the STZ-induceddiabetes and nonacupoint-acupunctured group (Fig. 1). Enhanced expression of nNOS in the DL-PAG was observed in the STZ-induced diabetic rats, and stimulation at the Zusanli acupoint significantly suppressed diabetesinduced enhancement of nNOS expression in the DL-PAG. A similar pattern was observed for NOS expression. The number of NADPH-d-positive cells in the control group was 448.21 ^ 14.24/mm 2, and the figure for the nondiabetic and Zusanli-acupunctured group and the nondiabetic and nonacupoint-acupunctured group was 499.24 ^ 15.79/mm 2 and 431.22 ^ 15.54/mm 2, respectively. This number was raised to 607.62 ^ 15.98/mm 2 in the STZ-induced diabetes group but was reduced again to 465.33 ^ 14.34/mm 2 in the STZinduced-diabetes and Zusanli-acupunctured group; the number was 587.00 ^ 16.54/mm 2 in the STZ-induceddiabetes and nonacupoint-acupunctured group (Fig. 2). Enhanced expression of NOS in the DL-PAG was observed
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in the STZ-induced diabetic rats, and stimulation at the Zusanli acupoint significantly suppressed diabetes-induced enhancement of NOS expression in the DL-PAG. In recently years, NO has been established as an important intercellular messenger; it has also been shown to act as
a messenger molecule in the augmentation of nociceptive transmission in the CNS and to induce hyperalgesia and allodynia [12]. Rodella et al. [16] reported that nociceptive visceral stimulation increases NOS activity in the rat PAG, and enhanced NOS immunoreactivity was observed in the
Fig. 1. Effect of acupuncture on nNOS expression in DL-PAG area. Upper: Photomicrographs of nNOS-positive cells in DL-PAG. Sections were stained for nNOS (reddish brown). Scale bar represents 100 mm. Lower: Mean number of nNOS-positive cells in the DL-PAG in each group. (A) Control group, (B) nondiabetic and Zusanli-acupunctured group, (C) nondiabetic and nonacupointacupunctured group, (D) STZ-induced-diabetes group, (E) STZinduced-diabetes and Zusanli-acupunctured group, (F) STZinduced-diabetes and nonacupoint-acupunctured group. *represents P , 0:05 compared to the control group. #represents P , 0:05 compared to the STZ-induced-diabetes group.
Fig. 2. Effect of acupuncture on NOS expression in DL-PAG area. Upper: Photomicrographs of NADPH-d-positive cells in the DLPAG. Sections were stained for NOS (blue). Scale bar represents 100 mm. Lower: Mean number of NADPH-d-positive cells in the DL-PAG in each group. (A) Control group, (B) nondiabetic and Zusanli-acupunctured group, (C) nondiabetic and nonacupointacupunctured group, (D) STZ-induced-diabetes group, (E) STZinduced-diabetes and Zusanli-acupunctured group, (F) STZinduced-diabetes and nonacupoint-acupunctured group. *represents P , 0:05 compared to the control group. #represents P , 0:05 compared to the STZ-induced-diabetes group.
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dorsal root ganglia in rats with neuropathic pain [18]. In addition, administration of 7-nitroindazole, a NOS inhibitor, was shown to have anti-nociceptive effect in mice [13], and constitutive NOS inhibitors were reported to potentiate the analgesic effect of morphine in tail-flick tests [15]. It was reported that PAG neurons constitute the primary center for anti-nociceptive action [20]. Neurons in the PAG of the midbrain are connected to the rostroventral medulla, and some neurons of the rostroventral medulla have inhibitory connections with neurons in the lamina of the spinal cord [19]. NO is known to inhibit neuronal activity in the PAG [11], and nociceptive visceral stimulation induces NOS expression in the PAG [16]. Acupunctural treatment, on the other hand, is known to activate PAG neurons [10]. It was reported that rats with STZ-induced diabetes suffer from allodynia and hyperalgesia [8]. In the present results, increased expression of nNOS and NOS was observed in the DL-PAG of STZ-induced diabetic rats, providing physiological evidence of the involvement of NOS in the allodynia and hyperalgesia in diabetic neuropathy. Acupuncture has been used for controlling various kinds of pain including lower back pain, chronic elbow pain, and toothache. Acupuncture-induced analgesia has been studied in patients with diabetic neuropathy and neuropathic animal models [6,8]. In the present results, acupunctural treatment at Zusanli was shown to exert no significant effect on the expression of nNOS and NOS in the DL-PAG under normal conditions while it suppressed the diabetes-induced enhancement in the expression of nNOS and NOS in the DL-PAG. The present results demonstrated that acupunctural treatment is effective in the modulation of the expression of the nNOS and NOS in the DL-PAG under diabetic conditions.
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[17] [1] Basbaum, A.I. and Fields, H.L., Endogenous pain control system: brain stem spinal pathways and endorphin circuitry, Annu. Rev. Neurosci., 7 (1984) 309–338. [2] Chang, S.L., Lin, J.G., Chi, T.C., Liu, I.M. and Cheng, J.T., An insulin-dependent hypoglycaemia induced by electroacupuncture at the Zhongwan (CV12) acupoint in diabetic rats, Diabetologia, 42 (1999) 250–255. [3] Courteix, C., Eschalier, A. and Lavarenne, J., Streptozotocin-induced diabetic rats: behavioural evidence for a model of chronic pain, Pain, 53 (1993) 81–88. [4] Dawson, T.M., Dawson, V.L. and Snyder, S.H., A novel neuronal messenger molecule in brain: the free radical, nitric oxide, Ann. Neurol., 32 (1992) 297–311. [5] Fox, A., Eastwood, C., Gentry, C., Manning, D. and Urban,
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L., Critical evaluation of the streptozotocin model of painful diabetic neuropathy in the rat, Pain, 81 (1999) 307–316. Goodnick, P.J., Breakstone, K., Wen, X.L. and Kumar, A., Acupuncture and neuropathy, Am. J. Psychiatry, 157 (2000) 1342–1343. Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811–2814. Hwang, B.G., Min, B.I., Kim, J.H., Na, H.S. and Park, D.S., Effects of electroacupuncture on the mechanical allodynia in the rat model of neuropathic pain, Neurosci. Lett., 320 (2002) 49–52. Kim, E.H., Jang, M.H., Shin, M.C., Lim, B.V., Kim, H.B., Kim, Y.J., Chung, J.H. and Kim, C.J., Acupuncture increases cell proliferation and neuropeptide Y expression in dentate gyrus of streptozotocin-induced diabetic rats, Neurosci. Lett., 327 (2002) 33–36. Lee, J.H. and Beitz, A.J., The distribution of brain stem and spinal cord nuclei associated with different frequencies of electroacupuncture analgesia, Pain, 52 (1993) 11–28. Lovick, T.A. and Key, B.J., Inhibitory effect of nitric oxide on neuronal activity in the periaqueductal grey matter of the rat, Exp. Brain Res., 108 (1996) 382–388. Meller, S.T. and Gebhart, G.F., Nitric oxide (NO) and nociceptive processing in the spinal cord, Pain, 52 (1993) 127– 136. Moore, P.K., Babbedge, R.C., Wallace, P., Gaffen, Z.A. and Hart, S.L., 7-Nitroindazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure, Br. J. Pharmacol., 108 (1993) 296–297. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, CA, 1997. Przewlocki, R., Machelska, H. and Przewlocki, B., Inhibition of nitric oxide synthase enhances morphine antinociception in rat spinal cord, Life Sci., 53 (1993) 1–5. Rodella, L., Rezzani, R., Agostini, C. and Bianchi, R., Induction of NADPH-diaphorase activity in the rat periaqueductal gray matter after nociceptive visceral stimulation, Brain Res., 793 (1998) 333–336. Sato, A., Sato, Y., Suzuki, A. and Uchida, S., Neural mechanisms of the reflex inhibition and excitation of gastric motility elicited by acupuncture-like stimulation in anesthetized rats, Neurosci. Res., 18 (1993) 53–62. Steel, J.H., Terenghi, G., Chung, J.M., Na, H.S., Carlton, S.M. and Polak, J.M., Increased nitric oxide synthase immunoreactivity in rat dorsal root ganglia in a neuropathic pain model, Neurosci. Lett., 169 (1994) 81–84. Takeshige, C., Sato, T., Mera, T., Hisamitsu, T. and Fang, J., Descending pain inhibitory system involved in acupuncture analgesia, Brain Res. Bull., 29 (1992) 617–634. Waters, A.J. and Lumb, B.M., Inhibitory effects evoked from both the lateral and periaqueductal grey are selective for the responses of rat dorsal horn neurons, Brain Res., 752 (1997) 239–249.
Acupuncture decreases nitric oxide synthase expression in periaqueductal gray area of rats with streptozotocin-induced diabetes Mi-Hyeon Jang a, Min-Chul Shin a, Gyo-Sung Koo b, Choong-Yeol Lee b, Ee-Hwa Kim c, Chang-Ju Kim a,* a
Department of Physiology, College of Medicine, Kyung Hee University, #1 Hoigi-dong, Dongdaemoon-gu, Seoul 130-701, South Korea b Department of Physiology, College of Oriental Medicine, Kyungwon University, Sungnam, South Korea c Department of Meridianology, College of Oriental Medicine, Semyung University, Jechon, South Korea Received 4 October 2002; accepted 12 November 2002
Abstract Acupuncture has been used as a clinical treatment in Oriental medicine for various diseases including diabetes mellitus, one of the most common metabolic disorders in humans. In the present study, the effect of acupuncture on the expressions of neuronal nitric oxide synthase (nNOS) and nitric oxide synthase (NOS) in the dorsolateral periaqueductal gray (DL-PAG) area of rats with streptozotocin (STZ)-induced diabetes was investigated via nNOS immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. Enhanced expression of nNOS and NOS was detected in the DL-PAG of rats with STZ-induced diabetes, and acupunctural treatment at Zusanli acupoint suppressed the diabetes-induced enhancement in the expression of nNOS and NOS. The present results demonstrate that acupuncture is effective in the modulation of the expression of nNOS and NOS in the DL-PAG under diabetic conditions. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acupuncture; Diabetes; Nitric oxide synthase; Neuronal nitric oxide synthase; Dorsolateral periaqueductal gray
Acupuncture has been used as a clinical treatment for various diseases in Oriental medicine. Acupuncture is known to possess many effects such as promotion of homeostasis, improvement in brain circulation, and pain control [8,17]. In addition, it has been reported that electroacupunctural stimulation is effective in treating diabetes [2,9]. Diabetes mellitus is one of the most common metabolic disorders in humans. In addition to the diabetic condition itself, numerous secondary complications are associated with the illness. It has been established that streptozotocin (STZ)-induced diabetes in rats produces a condition similar to the clinical form of diabetes mellitus, with severe peripheral neuropathy and inflammation [5]. Courteix et al. [3] reported that induction of insulin-dependent diabetes mellitus in rats causes allodynia and hyperalgesia. Nitric oxide (NO), synthesized from l-arginine through calcium-dependent pathways by nitric oxide synthase * Corresponding author. Tel.: 182-2-961-0407; fax: 182-2-9642195. E-mail address: [email protected] (C.-J. Kim).
(NOS), is a free radical with signaling functions in the central nervous system (CNS). NO has been implicated in the regulation of autonomic functions, and it has been shown to play important roles in the neural, vascular, and immune systems [4]. Several isoforms of NOS exist and fall into three major classes: inducible NOS, endothelial NOS, and neuronal NOS (nNOS). Of these, nNOS is mainly expressed in the CNS and has been implicated in signal transmission [4,7]. The midbrain periaqueductal gray (PAG) is believed to be an important component in the endogenous pain control system [1]. In immunohistochemical studies, the presence of NOS-staining neurons has been demonstrated in the dorsolateral PAG (DL-PAG). In the present study, the effect of acupuncture at the Zusanli acupoint (ST36) on the expression of nNOS and NOS in the DL-PAG was investigated via nNOS immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, which takes advantage of the fact that NADPH-d-positive neurons are the same as those containing NOS [7].
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S03 04 -3 94 0( 02 ) 01 318 - 6
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Male Sprague–Dawley rats weighing 200 ^ 10 g (6 weeks of age) were used for the experiment. Each animal was housed at a controlled temperature (20 ^ 2 8C) and was maintained under light–dark cycles, each cycle consisting of 12 h of light and 12 h of darkness (lights on from 07:00 h to 19:00 h), with food and water made available ad libitum. The experimental procedures were performed in accordance with the animal care guidelines of the NIH and the Korean Academy of Medical Sciences. Animals were divided into six groups: the control group, the nondiabetic and Zusanliacupunctured group, the nondiabetic and nonacupointacupunctured group, the STZ-induced-diabetes group, the STZ-induced-diabetes and Zusanli-acupunctured group, and the STZ-induced-diabetes and nonacupoint-acupunctured group (n ¼ 5 in each group). To induce diabetes in the experimental animals, a single intraperitoneal injection of STZ (50 mg/kg in saline; Sigma Chemical Co., St. Louis, MO) was given to each animal on the first day of the experiment, while animals of the control group and the nondiabetic groups received equivalent amounts of normal saline. Blood glucose levels were determined 2 days after STZ injection using a blood glucose analyzer (Arkray, Kyoto, Japan). Only the animals with blood glucose levels of 300 mg/dl or higher were used in this study. In the acupunctured groups, acupunctural treatment was given to each animal twice daily (10:00 a.m. and 5:00 p.m.) for 5 days, starting on the third day of the experiment. For acupunctural stimulation at Zusanli, stainless acupuncture needles of 0.3 mm diameter were bilaterally inserted into the locus of Zusanli, located 5 mm lateral and distal to the anterior tubercle of the tibia, and left in place for 20 min [9]. For treatment of animals of the nonacupoint-acupunctured groups, needles of the same diameter were inserted bilaterally at the hip and left in place for 20 min. All animals were sacrificed on the 7th day of the experiment. Animals were weighed and overdosed with Zoletil 50 w (10 mg/kg, i.p.; Vibac Laboratories, Carros, France). After a complete lack of response was observed, the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS) and then with 4% paraformaldehyde in 100 mM phosphate buffer (PB) at pH 7.4. The brains were dissected, postfixed in the same fixative overnight, and transferred into a 30% sucrose solution for cryoprotection. Serial coronal sections of 40 mm thickness were made using a freezing microtome (Leica, Nussloch, Germany). For analyzing the level of nNOS expression, ten sections on average were selected from each brain in the region spanning from Bregma 25.30– 2 8.30 mm according to the atlas by Paxinos and Watson [14]. Free-floating tissue sections were washed twice in 50 mM PBS and were then permeabilized in 0.2% Triton X-100 for 30 min. After washing twice with PBS, sections were incubated overnight with mouse anti-nNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:1000. Sections were washed twice in PBS and incubated for 1 h with biotinylated anti-rabbit antibody (1:200). Bound secondary antibody was
then amplified with Vector Elite ABC kit (Vector Laboratories, Burlingame, CA). The antibody-biotin-avidin-peroxidase complexes were visualized using 0.05% diaminobenzidine. The sections were mounted onto gelatinized glass slides and air-dried, and cover slides were mounted using Permount w. For NADPH-d activity, free-floating sections were incubated at 378C for 60 min in 100 mM PB containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 0.1 mg/ ml b-NADPH. The sections were then washed three times with PBS and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount w. The area of PAG was measured using Image-Pro wPlus image analyzer (Media Cybernetics Inc., Silver Spring, MD). The total numbers of nNOS-positive and NADPHd-positive neurons in the PAG were counted hemilaterally under a light microscope (Olympus, Tokyo, Japan), and the results were expressed as numbers of nNOS-positive and NADPH-d-positive cells per mm 2 of the area of the PAG region. Data was analyzed using SPSS by one-way analysis of variance followed by Scheffe´ ’s post-hoc test, and results were expressed as mean ^ standard error mean (SEM). Differences were considered significant for P , 0:05. In the present study, nNOS-positive and NADPH-d-positive cells were mainly localized in the dorsolateral area of the PAG, consistent with the findings of Rodella et al. [16]. The number of nNOS-positive cells in the animals of the control group (which received neither STZ nor acupunctural treatment) was 365.89 ^ 12.10/mm 2, and the figure for the nondiabetic and Zusanli-acupunctured group and the nondiabetic and nonacupoint-acupunctured group was 372.29 ^ 15.34/mm 2 and 342.11 ^ 4.43/ mm 2, respectively. This number was increased to 468.12 ^ 5.35/ mm 2 in the STZ-induced diabetes group (the animals of which did not receive any acupunctural treatment) but was decreased again to 370.43 ^ 6.08/mm 2 in the STZinduced-diabetes and Zusanli-acupunctured group; the number was 443.00 ^ 12.37/mm 2 in the STZ-induceddiabetes and nonacupoint-acupunctured group (Fig. 1). Enhanced expression of nNOS in the DL-PAG was observed in the STZ-induced diabetic rats, and stimulation at the Zusanli acupoint significantly suppressed diabetesinduced enhancement of nNOS expression in the DL-PAG. A similar pattern was observed for NOS expression. The number of NADPH-d-positive cells in the control group was 448.21 ^ 14.24/mm 2, and the figure for the nondiabetic and Zusanli-acupunctured group and the nondiabetic and nonacupoint-acupunctured group was 499.24 ^ 15.79/mm 2 and 431.22 ^ 15.54/mm 2, respectively. This number was raised to 607.62 ^ 15.98/mm 2 in the STZ-induced diabetes group but was reduced again to 465.33 ^ 14.34/mm 2 in the STZinduced-diabetes and Zusanli-acupunctured group; the number was 587.00 ^ 16.54/mm 2 in the STZ-induceddiabetes and nonacupoint-acupunctured group (Fig. 2). Enhanced expression of NOS in the DL-PAG was observed
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in the STZ-induced diabetic rats, and stimulation at the Zusanli acupoint significantly suppressed diabetes-induced enhancement of NOS expression in the DL-PAG. In recently years, NO has been established as an important intercellular messenger; it has also been shown to act as
a messenger molecule in the augmentation of nociceptive transmission in the CNS and to induce hyperalgesia and allodynia [12]. Rodella et al. [16] reported that nociceptive visceral stimulation increases NOS activity in the rat PAG, and enhanced NOS immunoreactivity was observed in the
Fig. 1. Effect of acupuncture on nNOS expression in DL-PAG area. Upper: Photomicrographs of nNOS-positive cells in DL-PAG. Sections were stained for nNOS (reddish brown). Scale bar represents 100 mm. Lower: Mean number of nNOS-positive cells in the DL-PAG in each group. (A) Control group, (B) nondiabetic and Zusanli-acupunctured group, (C) nondiabetic and nonacupointacupunctured group, (D) STZ-induced-diabetes group, (E) STZinduced-diabetes and Zusanli-acupunctured group, (F) STZinduced-diabetes and nonacupoint-acupunctured group. *represents P , 0:05 compared to the control group. #represents P , 0:05 compared to the STZ-induced-diabetes group.
Fig. 2. Effect of acupuncture on NOS expression in DL-PAG area. Upper: Photomicrographs of NADPH-d-positive cells in the DLPAG. Sections were stained for NOS (blue). Scale bar represents 100 mm. Lower: Mean number of NADPH-d-positive cells in the DL-PAG in each group. (A) Control group, (B) nondiabetic and Zusanli-acupunctured group, (C) nondiabetic and nonacupointacupunctured group, (D) STZ-induced-diabetes group, (E) STZinduced-diabetes and Zusanli-acupunctured group, (F) STZinduced-diabetes and nonacupoint-acupunctured group. *represents P , 0:05 compared to the control group. #represents P , 0:05 compared to the STZ-induced-diabetes group.
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dorsal root ganglia in rats with neuropathic pain [18]. In addition, administration of 7-nitroindazole, a NOS inhibitor, was shown to have anti-nociceptive effect in mice [13], and constitutive NOS inhibitors were reported to potentiate the analgesic effect of morphine in tail-flick tests [15]. It was reported that PAG neurons constitute the primary center for anti-nociceptive action [20]. Neurons in the PAG of the midbrain are connected to the rostroventral medulla, and some neurons of the rostroventral medulla have inhibitory connections with neurons in the lamina of the spinal cord [19]. NO is known to inhibit neuronal activity in the PAG [11], and nociceptive visceral stimulation induces NOS expression in the PAG [16]. Acupunctural treatment, on the other hand, is known to activate PAG neurons [10]. It was reported that rats with STZ-induced diabetes suffer from allodynia and hyperalgesia [8]. In the present results, increased expression of nNOS and NOS was observed in the DL-PAG of STZ-induced diabetic rats, providing physiological evidence of the involvement of NOS in the allodynia and hyperalgesia in diabetic neuropathy. Acupuncture has been used for controlling various kinds of pain including lower back pain, chronic elbow pain, and toothache. Acupuncture-induced analgesia has been studied in patients with diabetic neuropathy and neuropathic animal models [6,8]. In the present results, acupunctural treatment at Zusanli was shown to exert no significant effect on the expression of nNOS and NOS in the DL-PAG under normal conditions while it suppressed the diabetes-induced enhancement in the expression of nNOS and NOS in the DL-PAG. The present results demonstrated that acupunctural treatment is effective in the modulation of the expression of the nNOS and NOS in the DL-PAG under diabetic conditions.
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