Research reportResponses of neuronal nitric oxide synthase expression in the brainstem to electroacupuncture Zusanli (ST 36) in rats
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)
Brainstem projections of aortic baroreceptor afferent fibers in the rat
Neurosci. Lett.
(1983)- et al.
Localization of neurons with baroreceptor input in the medial solitary nucleus by means of intracellular application of horseradish peroxidase in the cat
Neurosci. Lett.
(1982) - et al.
Sympathetic effects of manual and electrical acupuncture of the tsusanli knee point: comparison with the hoku hand point sympathetic effects
Exp. Neurol.
(1986) - et al.
The correlation between neuron counts and optical density of NADPH-diaphorase histochemistry in the rat striatum: a quantitative study
Brain Res.
(1994) - et al.
Induction of spinal cord neuronal nitric oxide synthase (NOS) after formalin injection in the rat hind paw
Neurosci. Lett.
(1996) - et al.
The inhibitory role of nitric oxide (NO) in the somatocardiac sympathetic C-reflex in anesthetized rats
Neurosci. Res.
(1995) - et al.
Central noradrenergic activity is responsible for nitroglycerin-induced cardiovascular effects in the nucleus tractus solitarii
Brain Res.
(1991) - et al.
Responses of nitric oxide synthase expression in the gracile nucleus to sciatic nerve injury in young and aged rats
Brain Res.
(2000) - et al.
Barosensitive and chemosensitive neurons in the rat medulla: a double labeling study with c-Fos/glutamate, GAD, PNMT and calbindin
J. Auton. Nerv. Syst.
(1996) - et al.
Histochemical mapping of nitric oxide synthase in the rat brain
Neuroscience
(1992)
Neuronal nitric oxide synthase is induced in spinal neurona by traumatic injury
Neuroscience
Effects of nitric oxide on sympathetic baroreflex transmission in the nucleus tractus solitarii and caudal ventrolateral medulla in cats
Neurosci. Lett.
Interaction of cardiovascular reflexes in circulatory control
Visceral nociceptive input into the ventral posterolateral nucleus of the thalamus: a new function for the dorsal column pathway
J. Neurophysiol.
Pelvic visceral input into the nucleus gracilis is largely mediated by the postsynaptic dorsal column pathway
J. Neurophysiol.
Nucleus gracilis: an integrator for visceral and somatic information
J. Neurophysiol.
The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for up-regulated neuronal nitric oxide synthase
J. Exp. Med.
Responses of neurons in the gracile nucleus of cats to innocuous and noxious stimuli: basic characterization and antidromic activation from the thalamus
J. Neurophysiol.
l-glutamate evokes the release of an endothelium-derived relaxing factor-like substance from the rat nucleus tractus solitarius
J. Cardiovasc. Pharmacol.
The mechanisms of acupuncture analgesia
Br. J. Hosp. Med.
Neuronal responsiveness in the ventrobasal thalamic complex of rats with an experimental peripheral mononeuropathy
J. Neurophysiol.
The effect of spinal transection on acupuncture analgesia and morphine analgesia
Kexue Tongbao
Vasodilation induced by transcutaneous nerve stimulation in peripheral ischemia (Raynaud's phenomenon and diabetic polyneuropathy)
Eur. Heart J.
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