J Acupunct Meridian Stud 2008;1(1):4250

RE S E A R CH A RTI CL E

A Neurovascular Transmission Model for Acupuncture-induced Nitric Oxide

Sheng-Hsiung Hsiao1*, Li-Jen Tsai2
1 2

Division of Informatics, National Research Institute of Chinese Medicine, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

Received: Feb 29, 2008 Accepted: Apr 14, 2008 KEY WORDS:
acupuncture point; mechanotransduction; meridian; neurovascular transmission; nitric oxide; resonance theory

Abstract Acupuncture is the practice of inserting needles into the body to reduce pain or induce anesthesia. More broadly, acupuncture is a family of procedures involving the stimulation of anatomical locations on or in the skin by a variety of techniques. Employing acupuncture to treat human disease or maintain bodily condition has been practiced for thousands of years. However, the mechanism(s) of action of acupuncture at the various meridians are poorly understood. Most studies have indicated that acupuncture is able to increase blood flow. The acupuncture points have high electrical conductance and a relationship of the acupuncture points and meridians with the connective tissue planes and the perivascular space has also been suggested. Several studies employing the human and animal models have shown that acupuncture enhances the generation of nitric oxide (NO) and increases local circulation. Specifically, electroacupuncture (EA) seems to prevent the reduction in NO production from endothelial NO synthetase (eNOS) and neuronal NO synthase (nNOS) that is associated with hypertension and this process involves a stomachmeridian organ but not a non-stomach-meridian organ such as the liver. How can we explain the phenomena of EA and meridian effect? Here, we proposed a neurovascular transmission model for acupuncture induced NO. In this proposed model, the acupuncture stimulus is able to influence connective tissue via mechanical force transfer to the extracellular matrix (ECM). Through the ECM, the mechanotransduction stimulus can be translated or travel from the acupuncture points, which involve local tissue and cells. Cells in the local tissue that have received mechanotransduction induce different types of NO production that can induce changes in blood flow and local circulation. The local mechanical stress produced is coupled to a cyclic strain of the blood vessels and this could then change the frequency of resonance. According to the resonance theory, an oscillatory pattern of NO formation might occur in that specific organ. Therefore, the artery tree would then change the blood distribution and microcirculation of various organs and as a result further affect the production of NO.

*Corresponding author. National Research Institute of Chinese Medicine, No. 155-1, Section 2, Linong Street, Beitou District, Taipei 112, Taiwan. E-mail: leohsiao@nricm.edu.tw 2008 Korean Pharmacopuncture Institute

Neurovascular transmission model for acupuncture-induced NO

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1. Introduction
Acupuncture is the practice of inserting needles into the body to reduce pain or induce anesthesia. More broadly, acupuncture is a family of procedures involving the stimulation of anatomical locations on or in the skin by a variety of techniques. Employing acupuncture to treat human disease or to maintain bodily condition has been practiced for thousands of years. Recently, models able to describe the mode of action of acupuncture have aroused scientists curiosity. Scientific interest in acupuncture has led numerous investigators to conduct clinical trials that have tested the efficacy of acupuncture using various acupuncture points; however, the mechanism(s) of action of acupuncture at the various meridians are still poorly understood. Previous studies have provided a variety of information regarding the physiological effects of acupuncture on animal and human bodies. Most of them have indicated that acupuncture is able to increase blood flow [1] and that the acupuncture points and meridians have a high electrical conductance [2,3]. A relationship has also been suggested between acupuncture points and meridians with connective tissue planes [4] and the perivascular space [5]. A number of possible mechanisms by which acupuncture acts have been reviewed [6] and these results are available at the site of the National Center for Complementary and Alternative medicine (NCCAM) [7]. A Question and Answer page on How might acupuncture work? is also available on the website of the NCCAM at the National Institutes of Health where it states: It is proposed that acupuncture produces its effects through regulating the nervous system, thus aiding the activity of pain killing biochemicals such as endorphins and immune system cells at specific sites in the body. In addition, studies have shown that acupuncture may alter brain chemistry by changing the release of neurotransmitters and neurohormones and, thus, affecting the parts of the central nervous system related to sensation and involuntary body functions, such as immune reactions and processes that regulate a persons blood pressure, blood flow and body temperature. The results obtained from human and animal studies have also shown that acupuncture enhances the generation of nitric oxide (NO) and increases local circulation [8]. Kim et al demonstrated that employing acupuncture on stomach 36 point (ST36) is able to reduce blood pressure by activating NO signaling mechanisms [9]. Ma showed that NO content and NO synthase (NOS) expression were consistently higher at skin acupuncture points/meridians [10]. Chen et al showed that L-arginine-derived NO synthesis appears to mediate the noradrenergic

function of skin sympathetic nerve activation and that this contributes to skin electrical resistance of the acupuncture points and meridians [11]. NO is known to exert an effect on a number of functions including the regulation of blood pressure, contributing to the immune response, the control of neurotransmission and participation in cell differentiation and other physiological functions [12]. NO, a diffusible signaling gas, is synthesized by three NOS isoforms, namely a neuronal NOS (nNOS), an inducible NOS (iNOS) [13] and an endothelial NOS (eNOS) [14,15]. Using BH4 (tetrahydrobiopterin), Ca2+-calmodulin, Heme, flavin adenine dinucleotide, riboflavin monophosphate and NADPH [16] as cofactors and coupled with electron transfer, these enzymes are able to convert L-arginine to L-citruline and NO. Chinese acupuncture theory maintains that there are twelve main meridians or energy channels that relate to the internal organs. These include the lungs, large intestine, stomach, spleen, heart, the pericardium (the sac around the heart), etc. Electroacupuncture (EA) prevents the reduction of NO production from eNOS and nNOS associated with hypertension and has been shown to even increase eNOS and nNOS expression in stomach and cheek pouch tissue (which are also part of the stomach meridian) compared with a sham control. This effect was found not to occur on liver tissue, a nonstomach-meridian organ [9]. A question arises from the results of this study and this is how to explain the phenomena whereby EA was able to prevent the reduction of NO production from eNOS and nNOS associated with hypertension in a stomach-meridian organ but did not affect a non-stomach-meridian organ. Our hypothesis involves a neurovascular transmission model. Briefly, the acupuncture stimulus is able to induce a burst of NO production through mechanotransduction at the local acupuncture point and this NO diffuses and changes the blood flow either at the local and/or organ microcirculation level. The result of acupuncture is differential production of NO in various meridian organs, which are connected via tissue/cells coupled to the cyclically strained blood vessel; this is able to change the frequency of resonance.

2. A Neurovascular Transmission Model of Acupuncture

In the vascular wall, most of the bioavailable NO is believed to be derived from eNOS and diffuses into vascular smooth muscle and the blood stream, where it rapidly reacts with the hemoglobin (Hb) of the red blood cells. Hb, of course, is also able to transport oxygen and carbon dioxide. The blood

44 carries the oxygen complexed with Hb to all parts of the body where it is required for metabolism and also returns carrying carbon dioxide back to the lungs, where gaseous exchange occurs with the atmosphere. The peripheral chemoreceptors located in the carotid bodies respond primarily to hypoxemia. Central chemoreceptors located in the region of the brainstem respond to hypercapnia. Activation of either the hypoxic or hypercapnic chemoreflex elicits both hyperventilation and sympathetic activation [17]. There is evidence from animal and human studies that NO may play a role in hypercapnia induced vasodilation [18,19]. Recently, nNOS has been identified as a source of NO in the vicinity of microvessels and has been shown to participate in vascular function. Thus, NO can be produced and transported to the vascular smooth muscle cells from endothelial cells and perivascular nerve fibers, mast cells and other NOS-containing sources [20]. In Chinese terms, acupuncture is a healing art of inserting a needle into an acupuncture point in the meridian to correct an imbalance of Qi. The aim of acupuncture is to stimulate the flow of Qi through that meridian. To explain the meridian phenomenon, we proposed a neurovascular transmission model for acupuncture induced NO.

S.H. Hsiao, L.J. Tsai muscle and change blood flow and local circulation. L-arginine-derived NO synthesis appears to mediate the noradrenergic function that is part of skin sympathetic nerve activation and this contributes to the skin electrical resistance at acupuncture points and meridians [11].

2.3. The mechanical force of the needle coupled with cyclic strain of blood vessels changes the hemodynamic balance of the artery tree
Blood vessels are permanently subjected to mechanical forces in the form of stretching, which include cyclic mechanical strain. When acupunctured at a specific acupuncture point, the local mechanical stress produced is coupled with a cyclic strain of the blood vessel and this may change the resonance frequency. According to the resonance theory [24], a meridian can be classified according to its effects on the pulse spectrum and all meridian related effects, including those caused by acupuncture, are frequency specific. Therefore, the artery tree would change the blood distribution and microcirculation of various organs and further affect the production of NO by eNOS.

2.1. Acupuncture induced mechanotransduction

Immediately after a needle is inserted into connective tissue, the mechanical force is transferred to the extracellular matrix (ECM). The ECM is a multicomponent tissue that is able to transduce internal and external mechanical signals into changes in the tissue structure and function through a process termed mechanochemical transduction [21,22].

2.4. An oscillatory pattern of NO formation in a specific organ

For both agonist and hemodynamic stimuli, an oscillatory pattern of NO formation might occur with a burst of duration of a few seconds and a stimulidependent frequency in the order of tens of seconds to a few minutes [25]. Thus, by changing the hemodynamic state of the organ, an oscillatory pattern of NO formation might be created at a particular frequency in a specific organ [26]. In such a case, the frequency in the stomach ought to be different to that of the liver and therefore the NO production will also be different.

2.2. Mechanical force is able to change NO production, the blood vessels local circulation and skin sympathetic nerve activation
Through the ECM, the mechanical force stimulus can travel across the acupuncture point into the local tissue cells. The local tissue cells including arterioles, nerve terminals and mast cells can stimulate vascular nerve fibers; this will trigger nNOS and eNOS induced NO production [10]. Other tissues that may be involved include smooth muscle cells and endothelium cells; this is because the acupuncture is able to produce NO and this increases the blood flow, which change the local circulation [8,23]. Thus, mechanotransduction is able to trigger these cells to produce a burst of NO production, which diffuses into the vascular smooth

3. Evaluation of the Neurovascular Transmission Model of Acupuncture

Over the past 30 years, studies aimed at understanding the acupuncture point/meridian system have mainly looked for distinct histological features that might differentiate acupuncture points from the surrounding tissue. Several structures, such as neurovascular bundles [2729], neuromuscular attachments [3032] and various types of sensory nerve endings [33,34], have been described at acupuncture points.

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3.1. Mechanotransduction between the acupuncture point and connective tissue

In attempts to identify the anatomical and/or physiological properties of acupuncture points and meridians, various histological structures such as neurovascular bundles [2729] and various types of nerve endings [3036] have been reported at the acupuncture points. Skin impedance has also been found to be lower at acupuncture points compared with control points in some studies [37,38], but not in others [39,40]. Nevertheless, acupuncture points and meridians are frequently located along connective tissue planes that are between muscles or between muscle and bone or tendon [4143]. Traditional acupuncture theory proposes that the needling of appropriately selected acupuncture points has effects that are remote from the site of needle insertion and this is mediated by means of the meridian system [44]. To date, physiological models attempting to explain these remote effects have either invoked systemic mechanisms involving the nervous system [45,46], or involved signal transduction through connective tissue [45] together with the involvement of other systems including the nervous system. When needles are inserted into acupuncture points, the connective tissue around the acupuncture needle is wounded. This allows movement of the needle to pull and distort the surrounding tissue and this delivers a mechanical signal into the tissue at the cellular level [47,48]. Such mechanical signals are recognized as important mediators of information at the cellular level [49], can be transduced into bioelectrical and/or biochemical signals [50,51] and can lead to downstream effects, including cellular actin cytoskeleton polymerization, signaling pathway activation, changes in gene expression, protein synthesis and extracellular matrix modification [52,53]. Recent studies have suggested that both tissue stiffness and stress-induced electrical potentials are affected by the connective tissue matrix composition [54] and that changes in the matrix composition in response to mechanical stress may be an important form of communication between different cell types [55]. Changes in ECM composition can modulate the transduction of mechanical signals to, between and within cells [56]. Acupuncture needle manipulation, thus, may cause lasting modification of the ECM surrounding the needle, which may, in turn, influence the various cell populations sharing this connective tissue matrix such as fibroblasts, sensory afferents, immune and vascular cells. It has been suggested that the mechanism underlying needle grasp in acupuncture is caused by a muscle contraction [57,58]. The tissues involved

in needle grasp are the skin and/or subcutaneous connective tissues and the possible mechanisms for needle grasp involving these tissues include increased turgidity, contraction and the winding of tissue around the needle during needle rotation [59]. Because neurovascular bundles are located along connective tissue planes, the same amount of needle grasp may have more powerful downstream effects at acupuncture points due to the mechanical matrix deformation caused by tissue winding.

3.2. Acupuncture changes microcirculation via NO production

Acupuncture has been shown to improve cutaneous microcirculation and tissue healing in musculoskeletal flaps in rats [60,61] and to increase circulation in the skin above the parotid glands [62]. The mechanism underlying the increased blood flow resulting from acupuncture has been suggested to rely on the activation of thin nerve fibers and these release vasoactive neuropeptides and NO from their peripheral terminals upon activation, which leads to vasodilatation and increased blood flow [1,6365]. Traditionally, eNOS, which is primarily a membrane bound protein [66], is considered the principal source of bioavailable vascular NO. NO is produced by endothelial cells and diffuses into vascular smooth muscle and into the flowing blood, where it rapidly reacts with Hb in red blood cells. In recent years, the role of nNOS generated NO in vascular function has been clearly demonstrated [6769]. Kashiwagi et al [70] reported that nNOS containing nerve fibers, which innervate arterioles and nerve terminals, are the major sources of arteriolar NO, together with nNOS positive mast cells in rat mesentery and brain. Several other studies have reported nNOS to be present in perivascular nerve fibers [71].

3.3. Mechanical forces and cellular response

Blood vessels are continuously subjected to mechanical forces in the form of stretching and this encompasses cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, which then produce structural modifications of the arterial wall. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction and are able to transmit and modulate tension within the cell via focal adhesion

46 sites, integrins, cellular junctions and the ECM. Mechanical forces are also able to initiate complex signal transduction cascades, including the nuclear factor-kappaB and mitogen-activated protein kinase pathways and these lead to functional changes within the cell [72]. Mechanical forces, comprising both unidirectional laminar and oscillatory shear, are being recognized as important inducers of vascular NO generation. Laminar shear induces an increase in NO production through increased activation and expression of eNOS. Acutely, laminar shear activates eNOS through both Ca2+-dependent and Ca2+-independent mechanisms [73,74]. Oscillatory shear has also been shown to be able to stimulate an acute increase in NO production and upregulation of eNOS [75].

S.H. Hsiao, L.J. Tsai may be the same as the resonant frequency of its related internal organ. The resonance mechanism seems to be the key to understanding Chinese medicine if the unknown frequency of each meridian can be classified. From the pressure wave propagation equation, the resonance frequency of the organs (or tissues) main artery coupled system will decide the blood pressure energy distribution. Any physical disturbance to either the organ (tissue) or the artery will redistribute the blood pressure energy and therefore influence the blood distribution. Needling an acupoint is a disturbance that will cause an impedance mismatch and therefore influence the efficiency of the resonance. Stimulating an acupuncture point, which is a tissue rich with small arteries, is an effective way to cause the redistribution of blood pressure energy and therefore influence blood distribution. Thus, it has been shown that hemodynamic changes at the level of precapillary arterioles accompanying EA stimulation either of the hind paw or of the back mainly result in changes in the systemic arterial pressure regardless of stimulus current intensities [92]. From the above studies, we suggest that when acupuncture is applied at a specific acupuncture point, the local mechanical stress produced from needle grasp is able to trigger a burst of NO production and when this is coupled with the cyclic strain of the blood vessels, there is a change in the resonance frequency of blood vessels. This has an effect on the artery tree changing the various meridian organs blood distribution and microcirculation. This will have a further effect on the production of NO and NOS and create an amplification cycle.

3.4. A theoretical model involving an oscillatory pattern of NO induction and the resonance theory
Periodic bursts of intracellular free Ca2+ in response to a constant agonist concentration have been observed in a number of non-excitable cell types including endothelial cells [7678]. There is controversy regarding shear stress-induced Ca2+ transient release in endothelial cells with some investigators reporting multiple Ca2+ transient events [7982], whereas others report only their irregular appearance or none at all [8385]. Furthermore, both transient and sustained release of NO has been reported in response to shear stress. It is possible that shear stress regulates NO release in a Ca2+-independent fashion through phosporylation of eNOS, which results in a sustained basal NO production irrespective of the presence or absence of the Ca2+ transient effect [8688]. Kutchan and Frangos [89] measured the NO end oxidation products (NO2 and NO3) released from endothelial cells exposed to laminar flow and reported the presence of transient Ca2+dependent NO release at the onset of shear stress but continuous basal release in the presence of a constant shear stress level. Buerk and Riva [90] observed the presence of spontaneous low-frequency NO oscillations in the cat optic nerve head, which they attributed to a natural variation in shear stress. Kanai et al [91] measured NO release from endothelial cells exposed to constant shear stress using NO-sensitive microelectrodes and reported shear stress induced periodic Ca2+ transient release and a concomitant release of NO. A meridian is a hypothetical or functional line linking various arterial trees (acupuncture points) that have a similar resonance property [24]. In this weakly coupled system, there are two resonant frequencies at the acupuncture point and only one in the artery. The meridian selected frequency

4. Discussion
Acupuncture has been shown to improve cutaneous microcirculation and tissue healing in musculoskeletal flaps in rats [60,61] and to increase circulation in the skin above the parotid glands [62]. The mechanism underlying increased blood flow by acupuncture has been suggested to rely on the activation of thin nerve fibers, which release vasoactive neuropeptides and NO from their peripheral terminals upon activation; leading to vasodilatation and increased blood flow [1,63,64]. It is noted that because NO is involved in multiple body functions, its presence in the peripheral blood or in an acupuncture point is supportive of our hypothesis. To study the NO production from specific NOS, we can either block NO release by the use of a specific NO inhibitor or use specific NOS deficient mice. In general, it will be necessary to incorporate the interrelationship of the acupuncture points based on Chinese medicine principles into any such NO measurements.

Neurovascular transmission model for acupuncture-induced NO This will involve manipulating a distant point on the meridian and measure NO in another part of the body, or blocking NO in one part of the body and measure the inhibition by distant meridian manipulation. According to Traditional Chinese Medical theory, acupuncture points are functionally related to certain visceral organs and these relationships have been partially proven by clinical application of acupuncture therapy. Acupuncture point ST36 (stomach 36) is the most commonly used acupuncture point for the purpose of immune strengthening and immune regulation in oriental medical clinics (specifically strengthening and regulating Qi and blood in oriental medical terminology) [93]. Lees morphological study [94] suggest that there is a commonality of CNS cell groups in brain controlling the stomach (viscera) and the Zusanli point (ST36). A functional magnetic resonance imaging (fMRI) study was able to demonstrate that stimulation of ST36/SP6 specifically activated the orbital frontal cortex and de-activated the hippocampus [95]. Alternatively, stimulation of GB34/BL57 activated the dorsal thalamus and inhibited various primary motor areas and the premotor cortex. These results strongly support the existence of acupuncture point specificity. The modulatory effect of acupuncture on the cardiovascular and the sympathetic system has been referred to as somatoautonomic reflexes [96]. Manual acupuncture stimulation elicited a transient increase similar to skin sympathetic nerve activity (SSNA) and that this increase is dependent on the baseline of the SSNA [97]. EA can reduce sympathetic nerve activity (SNA) and arterial pressure (AP). Specifically, short term EA of ST36 (Zusanli) is able to reset the neural arc to a lower SNA, which moves the operating point toward lower AP and SNA under baroreflex closed loop conditions [98]. L-argininederived NO synthesis appears to mediate noradrenergic function during skin sympathetic nerve activation, which probably contributes to skin electrical resistance of the acupuncture points and meridians [11]. It has been speculated that acupuncture stimulates some sensory nerves or the autonomic nervous system and induces the recovery of blood circulation [99101]. NO content and nNOS expression have been shown to be consistently higher at the skin acupuncture points/meridians [10]. EA at ST-36 is regulated through NOS in organs on the stomach meridians [9]. ST-36 is used to promote blood flow and to treat cardiovascular disease; therefore, ST36 possibly induces its beneficial antihypertensive effect by activating NOS in the microcirculation. Meanwhile, acupuncturing ST-36 alone has been shown to produce different results when used to treat a renal hypertensive hamster compared with

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a sham-operated hamster. However, this is not universal for all acupuncture points and there was no response in organs that are not responsible for hypertension illness. According to Traditional Chinese Medical theory, when we look at pathway and disorder symptoms and compare the stomach meridian and the liver meridian, they differ in a number of aspects, but there is little data showing the differences in regulation between the organs. However, it is clear that the liver and stomach are located on separate meridian, each with its own mechanical signaling through connective tissue and as a result, the effects of acupuncture at a specific point are expressed differently. Here we propose a neurovascular transmission model starting from a local perspective and this model is able to suggest physiological processes that can bridge from the traditional nerve model of acupuncture to the vascular model. Based on this, we suggest that when studying organs located on and away from the same meridian as, for example, ST-36, it should be possible to identify the physiological mechanisms responsible for individual organ behavior. However, while acupuncturing ST-36 alone has produced different results between renal hypertensive hamster and sham-operated control [9], there is also a need to consider differences in the operation methods. Finally, we suggest that the best approach to a physiological study of acupuncture point either alone or combined with other acupuncture points should involve a broad range of measurements in addition to studying NOS. The Traditional Chinese Medical theory says: Qi acts as the commander of blood and blood acts as the mother of Qi. This is a general description of the effects of Qi on blood and it can be expanded to include the ideas that Qi controls the blood, promotes blood production and circulation, but also raises the notion of blood conveying Qi and blood nourishing Qi. If we suppose the NO is Qi, then the model we propose can be annotated as this part of Traditional Chinese Medical theory.

Acknowledgment
We thank Dr KW Liao and Dr WF Hsiaos comments on this manuscript.

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Neurovascular transmission model for acupuncture-induced NO

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