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Electroacupuncture decreases nitric oxide synthesis in the hypothalamus of spontaneously hypertensive rats
Neuroscience Letters 446 (2008) 78–82
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Electroacupuncture decreases nitric oxide synthesis in the hypothalamus of spontaneously hypertensive rats Jong-In Kim a , Yong-Suk Kim b , Sung-Keel Kang b , Changhwan Kim b , Chan Park c , Myeong Soo Lee a , Youngbuhm Huh c,∗ a
Department of Medical Research, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyunghee University, Seoul 130-701, Republic of Korea Department of Anatomy and Neurobiology, Biomedical Science Institute, School of Medicine, Kyunghee University, Hoeki-Dong 1, Dongdaemun-Gu, Seoul 130-701, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 11 July 2008 Received in revised form 2 September 2008 Accepted 17 September 2008 Keywords: Electroacupuncture Hypothalamus Neuronal nitric oxide synthase NADPH-diaphorase
a b s t r a c t Acupuncture-related effects on autonomic function have been explored via biological and neurophysiologic studies. The hypothalamus, known to regulate the autonomic nervous system, is likely affected by acupuncture treatment that modulates sympathetic functions. The aim of this study was to investigate the effect of electroacupuncture at the Jogsamni point (ST36, an acupoint known to modulate autonomic function) on expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus of spontaneously hypertensive rat. Nitric oxide, which is produced by nNOS activity, plays an important role in the regulation of many physiologic processes, including sympathetic activities, in the hypothalamus and other parts of the brain. nNOS expression was assessed by immunohistochemistry of nNOS and histochemistry of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). The staining intensities of nNOS-positive neurons and NADPH-d-positive neurons were quantitatively assessed using microdensitometry to measure changes in optical density. The results show that electroacupuncture at ST36 reduced the expression and activity of nNOS in the hypothalamus of spontaneously hypertensitive rats. These findings suggest that the electroacupuncture at ST36 results in modulation of the activity of nNOS in the hypothalamus of spontaneously hypertensive rat. © 2008 Elsevier Ireland Ltd. All rights reserved.
Acupuncture has been suggested as an effective method to maintain homeostasis, improve brain circulation, and control pain. Experimental and clinical studies suggest that afferent input via somatic nerve fibers, which may be induced by acupuncture, significantly influences pain control [5] as well as sympathetic functions and hormone production [2]. Acupuncture at analgesic acupoints, such as LI4, ear acupoint, P6 and ST 36 has also been shown to affect sympathetic functions, such as contraction of the urinary bladder, excretion of urine [14], modulation of heart function [12,18], and anti-inflammatory reactions [2] related to the autonomic nervous system. Although several neuroimaging studies [17,23] have described neural correlations between the hypothalamus and acupuncture stimulation at ST36, the molecular basis of this relationship remains unknown. Nitric oxide (NO) plays an important role in the regulation of many physiologic processes coordinated by the brain, including the regulation of autonomic functions, such as hypertension and
∗ Corresponding author. Tel.: +82 2 961 0285; fax: +82 2 968 1506. E-mail address: [email protected] (Y. Huh). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.09.049
stress [10]. Hypothalamic neurons that produce NO are primarily found in the paraventricular and supraoptic nuclei, although they are also found scattered in the dorsomedial, ventromedial, and lateral hypothalamus [22]. Over the past few decades, it has become clear that centrally produced NO is involved in the regulation of sympathetic signaling to the periphery [10,19]. Inhibition of central NO production increases sympathetic activity, while administration of l-arginine into several types of brain nuclei, such as the nucleus tractus solitarii and rostral ventrolateral medulla, reduce such activity [4,21]. These findings indicate that NO may affect sympathetic activity at hypothalamic regions in the central nervous system. In spontaneously hypertensive rat (SHR), the activity of neuronal nitric oxide synthase (nNOS) is increased owing to heightened sympathetic activity [19]. Acupuncture at ST36 has been known to affect sympathetic function [3]. Therefore, we hypothesized that acupuncture may alter sympathetic activity by decreasing nNOS activity in the hypothalamus. Thus, in the present study, we investigated the effect of electroacupuncture at ST36 on the expression of nNOS in the hypothalamus in SHR using immunohistochemistry of nNOS and histochemistry of nicotinamide adenine dinucleotide phosphate-
J.-I. Kim et al. / Neuroscience Letters 446 (2008) 78–82
diaphorase (NADPH-d). The latter procedure takes advantage of the fact that NADPH-d-positive neurons also contain nNOS. Twenty 4-week-old male SHR (Samtako Co., Korea) weighing 280–310 g were used in this study. The rats were acclimated for 2 weeks in cages at 21 ◦ C and provided with water and food ad libitum. The animals were randomly divided into an electroacupuncture group (n = 10) and a control group (n = 10). Animal experiments were carried out in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and experimental procedures were approved by the Institutional Animal Care and Use Committee at the Kyunghee University. The control group was kept under standard conditions without any stimulation. The electroacupuncture group was given electroacupuncture bilaterally at ST36 (Jogsamni, located 5 mm lateral and distal to the anterior tubercle of tibia), 3 times a week (total 10 times, during experimental weeks 3–5), under isoflurane anesthesia (in flow of oxygen and nitrous oxide mixture; 3% for induction and 1.5% for maintenance) in order to reduce the stress from electrical stimulation. Two stainless steel needles (Dongbang Co., Boryeong, Korea) with thickness of 0.15 mm were vertically inserted to a depth of 5 mm (cutaneous and muscle) at ST36 and at another point 10 mm inferior to ST36. Electroacupuncture was performed for 10 min. The positive electrode was connected to ST36, and the negative electrode to the inferior point. Electrical stimulation was provided by a pulse stimulator (Ito Co., Japan), which produced a biphasic square wave with the following characteristics: 2 Hz frequency, 1 mA, and 1 ms duration pulses. The current intensity was adjusted until localized muscle contractions were observed, and the polarity was reversed every 60 s to prevent polarization of the electrodes. After the termination of electroacupuncture, anesthesia was immediately discontinued and the rats resumed normal activity within 5 min. At 2 h after the last electroacupuncture treatment, SHRs in the control and electroacupuncture groups were anesthetized with pentobarbital sodium (60 mg/kg, i.p.) and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The brains of rats in each group were then removed, and frozen coronal sections with a thickness of 40 m were prepared. Sections were then stained for NADPH-d activity. Free-floating sections were incubated at 37 ◦ C for 60 min in 0.1 M PBS (pH 7.4) containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 1.0 mg/ml -NADPH. The sections were then washed three times with PBS and mounted on gelatin-coated slides. Following the three washes with PBS, the sections were processed for immunohistochemical detection of nNOS using mouse anti-nNOS antibody (Transduction Laboratories, Lexington, KY, USA). Free-floating sections were incubated for 48 h in PBS (4 ◦ C) containing anti-nNOS antiserum (1:1000 dilution), 0.3% Triton X100, 0.05% bovine serum albumin, and 1.5% normal horse serum. Using the Vectastain-Elite kit (Vector, Burlingame, CA, USA), sections were incubated for 1 h with biotinylated horse anti-mouse IgG secondary antibody (1:200), followed by incubation with avidin-biotin-peroxidase complex (1:100). Both incubations were carried out at room temperature. Sections were then treated with 0.02% 3,3 -diaminobenzidine tetrahydrochloride and 0.01% H2 O2 for 3 min. Staining of nNOS and NADPH-d was performed for every fifth section throughout the rostrocaudal extent of each rat from the electroacupuncture and control groups. Evaluation of the staining intensity of stained neurons was performed by measuring the optical density of nNOS- and NADPHd-positive neurons in 10 sections from the hypothalamic area. The optical density of the stained neurons was quantitatively assessed by microdensitometry using an image analyzer (Multiscan, Fullerton, USA). Before the measurement of densitometry,
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we evaluated the voltage-related change in optical density. Then, we obtained the optimal voltage from the linear portion of Sshaped voltage-related optical density curve [1]. During the full measurement of optical density, the optical voltage level was unchanged. Data are expressed as mean ± S.D. of the average optical density for each hypothalamic area. Statistical significance was estimated using the Student’s t-test, or Mann–Whitney U-test under the Shapiro-Wilk normality test (SAS program for Windows, version 9.1.3, USA). Differences were considered significant at P < 0.05. In the present study, nNOS- and NADPH-d-positive neurons were found to localize primarily in the paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus, and premammillary nucleus of the hypothalamus, consistent with the findings of Vincent and Kimura [22]. In the paraventricular nucleus, the optical density of nNOSpositive neurons in SHRs from the control and electroacupuncture groups was 145.6 ± 16.2 and 119.2 ± 13.8 arbitrary units (AU), respectively. In the supraoptic nucleus, the optical density of nNOS in the control and electroacupuncture groups was 165.9 ± 8.3 and 116.1 ± 8.8 AU, respectively. The optical density of nNOS in the ventromedial hypothalamus was 140.4 ± 9.0 AU in the control group and 109.1 ± 5.9 AU in the EA group. In the premammillary nucleus, the optical density of nNOS was 157.4 ± 11.4 AU in the control group and 122.5 ± 9.2 AU in the electroacupuncture group. Compared with the control group, the electroacupuncture group showed lower expression of nNOS throughout the examined hypothalamic nuclei (Fig. 1). A similar pattern was observed for NADPH-d stained sections (Fig. 2). In the paraventricular nucleus, the optical density of NADPH-d-positive neurons in the control group was 192.5 ± 21.0 AU, compared with 150.3 ± 20.8 AU for the electroacupuncture group. The optical density of NADPH-d in the supraoptic nucleus was 191.6 ± 16.2 and 149.2 ± 23.9 AU for the control and electroacupuncture groups, respectively. In the ventromedial hypothalamus, the optical density of NADPH-d was 173.5 ± 16.5 AU in the control group and 124.8 ± 21.4 AU in the electroacupuncture group. In the premammillary nucleus, the optical density of NADPH-d was 134.6 ± 21.8 AU in the control group and 76.9 ± 12.4 AU in the EA group. Because NADPH-d histochemistry correlates with NOS enzyme activity, these results indicate that electroacupuncture at ST36 reduces the activity of nNOS in the hypothalamus of SHRs. This study shows that electroacupuncture at ST36 decreases neuronal expression and activity of nNOS in the hypothalamus of SHR. The optical density of nNOS and NADPH-d-positive neurons decreased throughout the hypothalamic nuclei after electroacupuncture at ST36 in SHRs. The lateral hypothalamus projects onto the lateral medulla oblongata, where the cells comprising the autonomic nervous system are located. These cells include the parasympathetic vagal nuclei and a group of cells that descend to the sympathetic system in the spinal cord. It was shown more than a decade ago that the SHR exhibit increased sympathetic activity during the development of hypertension [15]. Previous studies have suggested that centrally produced NO is involved in the regulation of sympathetic outflow to the periphery, and thereby decreases arterial blood pressure [25]. Furthermore, gene expression of nNOS in the hypothalamus of adult SHR is increased in response to increased sympathetic activity [19]. Therefore, consistent with these previous studies, our data suggest that hypertension is related to the nNOS level in the hypothalamus of SHR. While it has been reported that electroacupuncture at ST36 inhibits nNOS expression at the brainstem in normotensive rat [16] and in the hypertensive rat [8], the neuronal change of nNOS induced by electroacupuncture at ST36 and the
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Fig. 1. Effect of electroacupuncture on nNOS in the hypothalamus in SHR. (A) Photomicrographs of nNOS-positive neurons in the control group (Cont) and the electroacupuncture group (EA): paraventricular nucleus (Pa), supraoptic nucleus (SO), ventromedial hypothalamic nucleus (VMH), and premammillary nucleus (PM). Sections were stained for nNOS (brown). The staining intensity of nNOS was lower in the hypothalamic nuclei of SHRs in the electroacupuncture group than in the control group. (B) Optical density of nNOS-positive neurons in the hypothalamus. The optical density was lower in the electroacupuncture group than in the control group. Ten animals were used in each group and data are expressed as mean ± S.D. * P < 0.0001 vs. control from a Student’s t-test, # P < 0.0001 vs. control from a Mann–Whitney U-test. Cont, control SHRs; EA, electroacupuncture SHRs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
J.-I. Kim et al. / Neuroscience Letters 446 (2008) 78–82
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Fig. 2. Effect of electroacupuncture on NADPH-d in the hypothalamus in SHR. (A) Photomicrographs of NADPH-d-positive neurons in the control group (Cont) and electroacupuncture group (EA): paraventricular nucleus (Pa), supraoptic nucleus (SO), ventromedial hypothalamic nucleus (VMH), and premammillary nucleus (PM). Sections were stained for NADPH-d (blue). The staining intensity of NADPH-d was lower in the hypothalamic nuclei of SHRs in electroacupuncture group than in the control group. (B) Optical density of NADPH-d-positive neurons in the hypothalamus. The optical density was lower in the electroacupuncture group than in the control group. Ten animals were used in each group and data are expressed as mean ± S.D. * P < 0.0001 vs. control from a Student’s t-test, # P < 0.0001 vs. control from a Mann–Whitney U-test. Cont, control SHRs; EA, electroacupuncture SHRs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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increased sympathetic activity in the hypothalamic region remain unexplained. Studies that employ combined staining for both nNOS and NADPH-d are necessary for a detailed examination of acupuncturerelated changes in nNOS-positive neurons in the rat brain. Examination of brain tissue using an anti-nNOS antibody can reveal relative levels of nNOS protein in different cells. Furthermore, NADPH-d histochemistry also correlates with nNOS activity. Kuo et al. [11] established a method for detecting quantitative changes using NADPH-d histochemistry, and several recent studies have used NADPH-d staining-related optical density as a measure of nNOS activity in the brain [6,24]. Therefore, optical density measurements of NADPH-d-positive neurons can reflect the relative levels of nNOS activity in fixed brain sections. In the present work, we found high intensity staining of NADPHd and nNOS in the hypothalamus of control SHRs, consistent with previous reports [7,19]. These previous findings led to speculation that increased sympathetic activity in hypertension may act as a stimulus for NO release in the hypothalamus. To that end, we found lower optical density of NADPH-d and nNOS staining in the hypothalamic nuclei of SHRs in the electroacupuncture group than in the control group SHRs. Hypothalamic neurons, which express nNOS, produce one or more neurohormones or neurotransmitters. In addition to NO, neurons located in the paraventricular and supraoptic nuclei also produce various other neurohormones and neurotransmitters, such as oxytocin, vasopressin, dopamine, corticotrophin-releasing hormone, and enkephalins. We suggest that the down-regulatory change of nNOS related with electroacupuncture may affect the synthesis and release of many of these neurohormones and neurotransmitters in the hypothalamus. The effect of acupuncture on the autonomic nervous system remains controversial both in humans and laboratory animals. Some groups have reported that electroacupuncture at LI4 has pressor effects on the dynamics of circulation, such as blood pressure, heart rate [13], muscle contraction [9], inflammation and renal nerve activity [14], by causing vasoconstriction via sympathetic outflow, or activation of sympathetic post-ganglionic neurons. In contrast, other studies support the inhibitory effect of acupuncture on the sympathetic nervous system. Sato et al. [20] reported that acupuncture in the perineal area inhibits efferent activities of the pudendal nerve-afferented pelvic nerve, and suppresses urinary bladder contraction. It has also been reported that the decrease in heart rate variability after acupuncture is the result of an increase in parasympathetic nerve activity and a decrease in sympathetic nerve activity in humans [12]. Therefore, along with the cited physiological findings of electroacupuncture, our results support the hypothesis that elecroacupuncture may attenuate sympathetic activity in the hypothalamus, the site of autonomic regulation. In conclusion, we found that electroacupuncture at ST36 effectively modulates the expression and activity of nNOS in the hypothalamus in SHR. We suggest that further research is necessary to identify specific neurotransmitters and neurohormones in other brain regions that are likely involved in modulation of sympathetic activity by electroacupuncture. Acknowledgement Jong-In Kim and Myeong Soo Lee received support from the Acupuncture, Moxibustion and Meridian Research Project (K08010) of Korea Institute of Oriental Medicine (KIOM).
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Ingram, The correlation between neuron counts and optical density of NADPH-diaphorase histochemistry in the rat striatum: a quantitative study, Brain Res. 660 (1994) 57–65. [12] Z. Li, C. Wang, A.F. Mak, D.H. Chow, Effects of acupuncture on heart rate variability in normal subjects under fatigue and non-fatigue state, Eur. J. Appl. Physiol. 94 (2005) 633–640. [13] J.M. Liao, H. Ting, S.D. Lee, C.H. Yang, Y.M. Liou, M.L. Peng, S.J. Tsai, C.F. Lin, T.B. Lin, Electroacupuncture-induced pressor and chronotropic effects in anesthetized rats, Auton. Neurosci. 124 (2006) 18–25. [14] T.B. Lin, T.C. Fu, C.F. Chen, Y.J. Lin, C.T. Chien, Low and high frequency electroacupuncture at Hoku elicits a distinct mechanism to activate sympathetic nervous system in anesthetized rats, Neurosci. Lett. 247 (1998) 155– 158. [15] S. Lundin, S.E. Ricksten, P. Thoren, Renal sympathetic activity in spontaneously hypertensive rats and normotensive controls, as studied by three different methods, Acta Physiol. 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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Electroacupuncture decreases nitric oxide synthesis in the hypothalamus of spontaneously hypertensive rats Jong-In Kim a , Yong-Suk Kim b , Sung-Keel Kang b , Changhwan Kim b , Chan Park c , Myeong Soo Lee a , Youngbuhm Huh c,∗ a
Department of Medical Research, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyunghee University, Seoul 130-701, Republic of Korea Department of Anatomy and Neurobiology, Biomedical Science Institute, School of Medicine, Kyunghee University, Hoeki-Dong 1, Dongdaemun-Gu, Seoul 130-701, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 11 July 2008 Received in revised form 2 September 2008 Accepted 17 September 2008 Keywords: Electroacupuncture Hypothalamus Neuronal nitric oxide synthase NADPH-diaphorase
a b s t r a c t Acupuncture-related effects on autonomic function have been explored via biological and neurophysiologic studies. The hypothalamus, known to regulate the autonomic nervous system, is likely affected by acupuncture treatment that modulates sympathetic functions. The aim of this study was to investigate the effect of electroacupuncture at the Jogsamni point (ST36, an acupoint known to modulate autonomic function) on expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus of spontaneously hypertensive rat. Nitric oxide, which is produced by nNOS activity, plays an important role in the regulation of many physiologic processes, including sympathetic activities, in the hypothalamus and other parts of the brain. nNOS expression was assessed by immunohistochemistry of nNOS and histochemistry of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). The staining intensities of nNOS-positive neurons and NADPH-d-positive neurons were quantitatively assessed using microdensitometry to measure changes in optical density. The results show that electroacupuncture at ST36 reduced the expression and activity of nNOS in the hypothalamus of spontaneously hypertensitive rats. These findings suggest that the electroacupuncture at ST36 results in modulation of the activity of nNOS in the hypothalamus of spontaneously hypertensive rat. © 2008 Elsevier Ireland Ltd. All rights reserved.
Acupuncture has been suggested as an effective method to maintain homeostasis, improve brain circulation, and control pain. Experimental and clinical studies suggest that afferent input via somatic nerve fibers, which may be induced by acupuncture, significantly influences pain control [5] as well as sympathetic functions and hormone production [2]. Acupuncture at analgesic acupoints, such as LI4, ear acupoint, P6 and ST 36 has also been shown to affect sympathetic functions, such as contraction of the urinary bladder, excretion of urine [14], modulation of heart function [12,18], and anti-inflammatory reactions [2] related to the autonomic nervous system. Although several neuroimaging studies [17,23] have described neural correlations between the hypothalamus and acupuncture stimulation at ST36, the molecular basis of this relationship remains unknown. Nitric oxide (NO) plays an important role in the regulation of many physiologic processes coordinated by the brain, including the regulation of autonomic functions, such as hypertension and
∗ Corresponding author. Tel.: +82 2 961 0285; fax: +82 2 968 1506. E-mail address: [email protected] (Y. Huh). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.09.049
stress [10]. Hypothalamic neurons that produce NO are primarily found in the paraventricular and supraoptic nuclei, although they are also found scattered in the dorsomedial, ventromedial, and lateral hypothalamus [22]. Over the past few decades, it has become clear that centrally produced NO is involved in the regulation of sympathetic signaling to the periphery [10,19]. Inhibition of central NO production increases sympathetic activity, while administration of l-arginine into several types of brain nuclei, such as the nucleus tractus solitarii and rostral ventrolateral medulla, reduce such activity [4,21]. These findings indicate that NO may affect sympathetic activity at hypothalamic regions in the central nervous system. In spontaneously hypertensive rat (SHR), the activity of neuronal nitric oxide synthase (nNOS) is increased owing to heightened sympathetic activity [19]. Acupuncture at ST36 has been known to affect sympathetic function [3]. Therefore, we hypothesized that acupuncture may alter sympathetic activity by decreasing nNOS activity in the hypothalamus. Thus, in the present study, we investigated the effect of electroacupuncture at ST36 on the expression of nNOS in the hypothalamus in SHR using immunohistochemistry of nNOS and histochemistry of nicotinamide adenine dinucleotide phosphate-
J.-I. Kim et al. / Neuroscience Letters 446 (2008) 78–82
diaphorase (NADPH-d). The latter procedure takes advantage of the fact that NADPH-d-positive neurons also contain nNOS. Twenty 4-week-old male SHR (Samtako Co., Korea) weighing 280–310 g were used in this study. The rats were acclimated for 2 weeks in cages at 21 ◦ C and provided with water and food ad libitum. The animals were randomly divided into an electroacupuncture group (n = 10) and a control group (n = 10). Animal experiments were carried out in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and experimental procedures were approved by the Institutional Animal Care and Use Committee at the Kyunghee University. The control group was kept under standard conditions without any stimulation. The electroacupuncture group was given electroacupuncture bilaterally at ST36 (Jogsamni, located 5 mm lateral and distal to the anterior tubercle of tibia), 3 times a week (total 10 times, during experimental weeks 3–5), under isoflurane anesthesia (in flow of oxygen and nitrous oxide mixture; 3% for induction and 1.5% for maintenance) in order to reduce the stress from electrical stimulation. Two stainless steel needles (Dongbang Co., Boryeong, Korea) with thickness of 0.15 mm were vertically inserted to a depth of 5 mm (cutaneous and muscle) at ST36 and at another point 10 mm inferior to ST36. Electroacupuncture was performed for 10 min. The positive electrode was connected to ST36, and the negative electrode to the inferior point. Electrical stimulation was provided by a pulse stimulator (Ito Co., Japan), which produced a biphasic square wave with the following characteristics: 2 Hz frequency, 1 mA, and 1 ms duration pulses. The current intensity was adjusted until localized muscle contractions were observed, and the polarity was reversed every 60 s to prevent polarization of the electrodes. After the termination of electroacupuncture, anesthesia was immediately discontinued and the rats resumed normal activity within 5 min. At 2 h after the last electroacupuncture treatment, SHRs in the control and electroacupuncture groups were anesthetized with pentobarbital sodium (60 mg/kg, i.p.) and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The brains of rats in each group were then removed, and frozen coronal sections with a thickness of 40 m were prepared. Sections were then stained for NADPH-d activity. Free-floating sections were incubated at 37 ◦ C for 60 min in 0.1 M PBS (pH 7.4) containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 1.0 mg/ml -NADPH. The sections were then washed three times with PBS and mounted on gelatin-coated slides. Following the three washes with PBS, the sections were processed for immunohistochemical detection of nNOS using mouse anti-nNOS antibody (Transduction Laboratories, Lexington, KY, USA). Free-floating sections were incubated for 48 h in PBS (4 ◦ C) containing anti-nNOS antiserum (1:1000 dilution), 0.3% Triton X100, 0.05% bovine serum albumin, and 1.5% normal horse serum. Using the Vectastain-Elite kit (Vector, Burlingame, CA, USA), sections were incubated for 1 h with biotinylated horse anti-mouse IgG secondary antibody (1:200), followed by incubation with avidin-biotin-peroxidase complex (1:100). Both incubations were carried out at room temperature. Sections were then treated with 0.02% 3,3 -diaminobenzidine tetrahydrochloride and 0.01% H2 O2 for 3 min. Staining of nNOS and NADPH-d was performed for every fifth section throughout the rostrocaudal extent of each rat from the electroacupuncture and control groups. Evaluation of the staining intensity of stained neurons was performed by measuring the optical density of nNOS- and NADPHd-positive neurons in 10 sections from the hypothalamic area. The optical density of the stained neurons was quantitatively assessed by microdensitometry using an image analyzer (Multiscan, Fullerton, USA). Before the measurement of densitometry,
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we evaluated the voltage-related change in optical density. Then, we obtained the optimal voltage from the linear portion of Sshaped voltage-related optical density curve [1]. During the full measurement of optical density, the optical voltage level was unchanged. Data are expressed as mean ± S.D. of the average optical density for each hypothalamic area. Statistical significance was estimated using the Student’s t-test, or Mann–Whitney U-test under the Shapiro-Wilk normality test (SAS program for Windows, version 9.1.3, USA). Differences were considered significant at P < 0.05. In the present study, nNOS- and NADPH-d-positive neurons were found to localize primarily in the paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus, and premammillary nucleus of the hypothalamus, consistent with the findings of Vincent and Kimura [22]. In the paraventricular nucleus, the optical density of nNOSpositive neurons in SHRs from the control and electroacupuncture groups was 145.6 ± 16.2 and 119.2 ± 13.8 arbitrary units (AU), respectively. In the supraoptic nucleus, the optical density of nNOS in the control and electroacupuncture groups was 165.9 ± 8.3 and 116.1 ± 8.8 AU, respectively. The optical density of nNOS in the ventromedial hypothalamus was 140.4 ± 9.0 AU in the control group and 109.1 ± 5.9 AU in the EA group. In the premammillary nucleus, the optical density of nNOS was 157.4 ± 11.4 AU in the control group and 122.5 ± 9.2 AU in the electroacupuncture group. Compared with the control group, the electroacupuncture group showed lower expression of nNOS throughout the examined hypothalamic nuclei (Fig. 1). A similar pattern was observed for NADPH-d stained sections (Fig. 2). In the paraventricular nucleus, the optical density of NADPH-d-positive neurons in the control group was 192.5 ± 21.0 AU, compared with 150.3 ± 20.8 AU for the electroacupuncture group. The optical density of NADPH-d in the supraoptic nucleus was 191.6 ± 16.2 and 149.2 ± 23.9 AU for the control and electroacupuncture groups, respectively. In the ventromedial hypothalamus, the optical density of NADPH-d was 173.5 ± 16.5 AU in the control group and 124.8 ± 21.4 AU in the electroacupuncture group. In the premammillary nucleus, the optical density of NADPH-d was 134.6 ± 21.8 AU in the control group and 76.9 ± 12.4 AU in the EA group. Because NADPH-d histochemistry correlates with NOS enzyme activity, these results indicate that electroacupuncture at ST36 reduces the activity of nNOS in the hypothalamus of SHRs. This study shows that electroacupuncture at ST36 decreases neuronal expression and activity of nNOS in the hypothalamus of SHR. The optical density of nNOS and NADPH-d-positive neurons decreased throughout the hypothalamic nuclei after electroacupuncture at ST36 in SHRs. The lateral hypothalamus projects onto the lateral medulla oblongata, where the cells comprising the autonomic nervous system are located. These cells include the parasympathetic vagal nuclei and a group of cells that descend to the sympathetic system in the spinal cord. It was shown more than a decade ago that the SHR exhibit increased sympathetic activity during the development of hypertension [15]. Previous studies have suggested that centrally produced NO is involved in the regulation of sympathetic outflow to the periphery, and thereby decreases arterial blood pressure [25]. Furthermore, gene expression of nNOS in the hypothalamus of adult SHR is increased in response to increased sympathetic activity [19]. Therefore, consistent with these previous studies, our data suggest that hypertension is related to the nNOS level in the hypothalamus of SHR. While it has been reported that electroacupuncture at ST36 inhibits nNOS expression at the brainstem in normotensive rat [16] and in the hypertensive rat [8], the neuronal change of nNOS induced by electroacupuncture at ST36 and the
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Fig. 1. Effect of electroacupuncture on nNOS in the hypothalamus in SHR. (A) Photomicrographs of nNOS-positive neurons in the control group (Cont) and the electroacupuncture group (EA): paraventricular nucleus (Pa), supraoptic nucleus (SO), ventromedial hypothalamic nucleus (VMH), and premammillary nucleus (PM). Sections were stained for nNOS (brown). The staining intensity of nNOS was lower in the hypothalamic nuclei of SHRs in the electroacupuncture group than in the control group. (B) Optical density of nNOS-positive neurons in the hypothalamus. The optical density was lower in the electroacupuncture group than in the control group. Ten animals were used in each group and data are expressed as mean ± S.D. * P < 0.0001 vs. control from a Student’s t-test, # P < 0.0001 vs. control from a Mann–Whitney U-test. Cont, control SHRs; EA, electroacupuncture SHRs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 2. Effect of electroacupuncture on NADPH-d in the hypothalamus in SHR. (A) Photomicrographs of NADPH-d-positive neurons in the control group (Cont) and electroacupuncture group (EA): paraventricular nucleus (Pa), supraoptic nucleus (SO), ventromedial hypothalamic nucleus (VMH), and premammillary nucleus (PM). Sections were stained for NADPH-d (blue). The staining intensity of NADPH-d was lower in the hypothalamic nuclei of SHRs in electroacupuncture group than in the control group. (B) Optical density of NADPH-d-positive neurons in the hypothalamus. The optical density was lower in the electroacupuncture group than in the control group. Ten animals were used in each group and data are expressed as mean ± S.D. * P < 0.0001 vs. control from a Student’s t-test, # P < 0.0001 vs. control from a Mann–Whitney U-test. Cont, control SHRs; EA, electroacupuncture SHRs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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increased sympathetic activity in the hypothalamic region remain unexplained. Studies that employ combined staining for both nNOS and NADPH-d are necessary for a detailed examination of acupuncturerelated changes in nNOS-positive neurons in the rat brain. Examination of brain tissue using an anti-nNOS antibody can reveal relative levels of nNOS protein in different cells. Furthermore, NADPH-d histochemistry also correlates with nNOS activity. Kuo et al. [11] established a method for detecting quantitative changes using NADPH-d histochemistry, and several recent studies have used NADPH-d staining-related optical density as a measure of nNOS activity in the brain [6,24]. Therefore, optical density measurements of NADPH-d-positive neurons can reflect the relative levels of nNOS activity in fixed brain sections. In the present work, we found high intensity staining of NADPHd and nNOS in the hypothalamus of control SHRs, consistent with previous reports [7,19]. These previous findings led to speculation that increased sympathetic activity in hypertension may act as a stimulus for NO release in the hypothalamus. To that end, we found lower optical density of NADPH-d and nNOS staining in the hypothalamic nuclei of SHRs in the electroacupuncture group than in the control group SHRs. Hypothalamic neurons, which express nNOS, produce one or more neurohormones or neurotransmitters. In addition to NO, neurons located in the paraventricular and supraoptic nuclei also produce various other neurohormones and neurotransmitters, such as oxytocin, vasopressin, dopamine, corticotrophin-releasing hormone, and enkephalins. We suggest that the down-regulatory change of nNOS related with electroacupuncture may affect the synthesis and release of many of these neurohormones and neurotransmitters in the hypothalamus. The effect of acupuncture on the autonomic nervous system remains controversial both in humans and laboratory animals. Some groups have reported that electroacupuncture at LI4 has pressor effects on the dynamics of circulation, such as blood pressure, heart rate [13], muscle contraction [9], inflammation and renal nerve activity [14], by causing vasoconstriction via sympathetic outflow, or activation of sympathetic post-ganglionic neurons. In contrast, other studies support the inhibitory effect of acupuncture on the sympathetic nervous system. Sato et al. [20] reported that acupuncture in the perineal area inhibits efferent activities of the pudendal nerve-afferented pelvic nerve, and suppresses urinary bladder contraction. It has also been reported that the decrease in heart rate variability after acupuncture is the result of an increase in parasympathetic nerve activity and a decrease in sympathetic nerve activity in humans [12]. Therefore, along with the cited physiological findings of electroacupuncture, our results support the hypothesis that elecroacupuncture may attenuate sympathetic activity in the hypothalamus, the site of autonomic regulation. In conclusion, we found that electroacupuncture at ST36 effectively modulates the expression and activity of nNOS in the hypothalamus in SHR. We suggest that further research is necessary to identify specific neurotransmitters and neurohormones in other brain regions that are likely involved in modulation of sympathetic activity by electroacupuncture. Acknowledgement Jong-In Kim and Myeong Soo Lee received support from the Acupuncture, Moxibustion and Meridian Research Project (K08010) of Korea Institute of Oriental Medicine (KIOM).
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