Elsevier

Brain Research

Volume 1037, Issues 1–2, 10 March 2005, Pages 70-77
Brain Research

Research report
Responses of neuronal nitric oxide synthase expression in the brainstem to electroacupuncture Zusanli (ST 36) in rats

https://doi.org/10.1016/j.brainres.2004.12.029Get rights and content

Abstract

Recent studies have reported that l-arginine-derived nitric oxide (NO) in the gracile nucleus modifies the hypotensive responses to electroacupuncture (EA) stimulation of Zusanli (ST 36). The purpose of this study was to examine the influence of EA stimulation of ST 36 on neuronal NO synthase (nNOS) expression in the brainstem nuclei in rats. EA stimulation of ST 36 and a non-acupoint was performed using 3 Hz of stimulation for 10 s every 2 min for a period of 120 min in rats anesthetized with ketamine. Rats in the sham-treated group received surgery and EA needles were placed into the acupoints without performing the stimulation. After 2-h stimulation and sham treatment, animals were perfused with 4% paraformaldehyde. Sections of rat medulla were examined by immunolabeling with a polyclonal antibody directed against nNOS. The brainstem nuclei were also visualized by NADPH-diaphorase histochemistry, a marker of nNOS activity. nNOS expression and NADPH-diaphorase reactivity were quantified by using a microscope with reticule grid to count the number of positive cells over a nucleus. Unilateral EA stimulation of ST 36 in rats caused increases in nNOS immunostained cells in the rostral region of the ipsilateral gracile nucleus, but was not altered in the contralateral gracile nucleus compared with sham-treated rats (P < 0.05, n = 6–7). NADPH-diaphorase-positive cells were also increased in the ipsilateral gracile nucleus of rats with EA stimulation. nNOS immunostaining and NADPH-diaphorase-positive neurons were significantly increased in both ipsilateral and contralateral sides of the medial nucleus tractus solitarius (mNTS) in rats receiving EA ST 36 compared with sham-treated animals (P < 0.05). nNOS immunostaining and NADPH-diaphorase reactivity was neither altered in the gracile nucleus and mNTS of non-acupoint stimulated rats nor other brainstem nuclei in rats with EA ST 36. These results show that nNOS immunoreactivity and NADPH-diaphorase reactivity are consistently increased in the gracile nucleus and the mNTS by EA ST 36. We conclude that EA ST 36 induces nNOS expression in the gracile nucleus and mNTS, and enhanced nNOS-NO in the nuclei may modify central cardiovascular regulation, which contribute to hypotensive effects of acupuncture.

Introduction

Acupuncture has been used for centuries in the treatment and prevention of various diseases, including cardiovascular diseases and diseases with hypertensive syndromes [10], [31]. Low-frequency electroacupuncture (EA) stimulation of lower extremity acupuncture point (acupoint) Zusanli (ST 36) in human [10] and low-frequency transcutaneous nerve stimulation [14], [15] induce long-lasting sympathoinhibition and widespread vasodilation. Investigators have demonstrated that the nervous system, neurotransmitters, and endogenous substances respond to EA stimulation [11], [13], [31], [36]. It has been established that acupuncture analgesia is mediated by opioid peptides in the brain [11], [13]. However, the mechanisms responsible for hypotensive responses to EA of ST 36 are unclear.

A number of studies have suggested that nitric oxide (NO) in the nucleus tractus solitarius (NTS) plays an important role in the central inhibition of sympathetic tone, thus decreasing arterial blood pressure [32], [38], [45]. Somatocardiac sympathetic C-reflex is also centrally inhibited by the presence of NO in the brainstem [21]. Recent experiments in rats have demonstrated that the gracile nucleus plays an inhibitory role in cardiovascular responses to stimulus-evoked somatosympathetic reflexes (SSR) in rats [34]. l-arginine-derived NO in the gracile nucleus modifies the arterial blood depressor responses to EA stimulation of ST 36 [35]. Neuronal NO synthase (nNOS) expression is also enhanced in the gracile nucleus by peripheral nerve stimulation evoked activation of SSR [24].

Electrophysiological mapping studies and anterograde axonal tracing studies have shown that peripheral somatosensory afferents projecting from the hindlimb to the medulla are distributed mainly in the gracile nucleus, and stimulation of somatic sensory nerves results in changes in sympathetic nerve activity and arterial blood pressure [7], [20], [33], [40]. A number of recent studies have suggested that the gracile nucleus is an integration center for cutaneous and visceral information flowing into the thalamus, which plays an important role in somatic and visceral pain processing [2], [3], [4]. Recent studies have also reported that sensory stimulation of the hindlimb somatic afferent modifies neuronal activities in the NTS [39]. It is well documented that NTS is the principal sensory nucleus for the central regulation of cardiovascular function through the NTS-rostral ventral medulla (RVM) central sympathetic pathways [1], [37]. There has been emphasis that the medial portion NTS (mNTS) is a region of particularly dense innervation and mediation of the cardiovascular reflex responses by physiological and anatomical studies [6], [8].

The purpose of the present study was to determine the influence of EA stimulation of lower limb acupoint Zusanli (ST 36) on nNOS expression in the medulla, and examine whether electrical stimulation of the acupoint enhanced nNOS expression in the brainstem nuclei, particularly in the mNTS and gracile nucleus, compared to stimulation of non-acupoint and sham-treated rats without the stimulation. nNOS expressions in the gracile nucleus, mNTS, and RVM were examined by using nNOS immunohistochemistry. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) reactivity, a marker of nNOS activity, was also investigated in the brainstem nuclei following the treatments.

Section snippets

Electroacupuncture stimulation method

All experiments were performed using adult (5–6 months old and 350–400 g) male Sprague–Dawley rats. The protocol was approved by the Harbor-UCLA Animal Care and Use Review Committee, and was in accord with AAALAC and NIH guidelines. The animals were maintained on a 12-h light–dark cycle in temperature and humidity controlled rooms. Food and water were available ad libitum.

The EA stimulation was performed in rats anesthetized with ketamine (100 mg/kg i.p.) plus xylazine (13 mg/kg, i.p.). The

NADPH-diaphorase staining

The levels of NADPHd reactivity in the gracile nucleus, mNTS, and RVM were examined in rats with EA stimulation (n = 7) of ST 36 compared to sham-treated rats (n = 7). Fig. 1 shows gracile nucleus and NTS neurons from a sham-treated and EA-treated rat that contain variable NADPHd reactivity as evidenced by the color density of the cells. In sham-treated groups, the gracile nucleus of rats exhibited light NADPHd staining of medium-sized neurons and a moderately dense nerve fiber network (Fig. 1,

Discussion

We examined the influence of unilateral EA stimulation applied to the hindlimb acupoint Zusanli (ST 36) on nNOS immunoreactivity and NADPHd reactivity in the brainstem of rats. The major new findings of this study are: (1) NADPHd reactivity and nNOS-positive neurons were significantly increased by EA of ST 36 in the ipsilateral side of the rostral region of the gracile nucleus, but not in the caudal gracile nucleus or in the contralateral side; (2) nNOS and NADPHd-positive cells were increased

Acknowledgments

This research was supported by NIH (AT00450, HL04447, AT002478, and HD39169) to SXM. These studies were conducted at the biomedical research facilities of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center.

References (46)

  • W. Wu et al.

    Neuronal nitric oxide synthase is induced in spinal neurona by traumatic injury

    Neuroscience

    (1994)
  • J. Zanzinger et al.

    Effects of nitric oxide on sympathetic baroreflex transmission in the nucleus tractus solitarii and caudal ventrolateral medulla in cats

    Neurosci. Lett.

    (1995)
  • F.M. Abboud et al.

    Interaction of cardiovascular reflexes in circulatory control

  • E.D. Al-Chaer et al.

    Visceral nociceptive input into the ventral posterolateral nucleus of the thalamus: a new function for the dorsal column pathway

    J. Neurophysiol.

    (1996)
  • E.D. Al-Chaer et al.

    Pelvic visceral input into the nucleus gracilis is largely mediated by the postsynaptic dorsal column pathway

    J. Neurophysiol.

    (1996)
  • E.D. Al-Chaer et al.

    Nucleus gracilis: an integrator for visceral and somatic information

    J. Neurophysiol.

    (1997)
  • A.R. Amin et al.

    The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for up-regulated neuronal nitric oxide synthase

    J. Exp. Med.

    (1995)
  • K.D. Cliffer et al.

    Responses of neurons in the gracile nucleus of cats to innocuous and noxious stimuli: basic characterization and antidromic activation from the thalamus

    J. Neurophysiol.

    (1992)
  • E.D. Di Paola et al.

    l-glutamate evokes the release of an endothelium-derived relaxing factor-like substance from the rat nucleus tractus solitarius

    J. Cardiovasc. Pharmacol.

    (1991)
  • J.M.G. Foster et al.

    The mechanisms of acupuncture analgesia

    Br. J. Hosp. Med.

    (1987)
  • G. Guilbaud et al.

    Neuronal responsiveness in the ventrobasal thalamic complex of rats with an experimental peripheral mononeuropathy

    J. Neurophysiol.

    (1990)
  • J.S. Han et al.

    The effect of spinal transection on acupuncture analgesia and morphine analgesia

    Kexue Tongbao

    (1986)
  • B. Kaada

    Vasodilation induced by transcutaneous nerve stimulation in peripheral ischemia (Raynaud's phenomenon and diabetic polyneuropathy)

    Eur. Heart J.

    (1982)
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