Stimuli-induced nitric oxide and neuropeptides mediated axon reflexes contribute to tracers along meridian pathways

Abstract

An abundance of studies from different international groups have demonstrated that tracers along linear pathways resembling meridians over the body surface of humans. All experiments of the studies have been conducted by injection of a radiotracer solution or tracer dyes in a volume of solution into acupuncture points (acupoints). Solution injected into acupoints produce much stronger mechanical stimuli than acupuncture, which cause axon reflex. Anatomical studies have demonstrated that acupoints/meridians exist higher number of small nerve fibers and blood vessels with rich nitric oxide (NO) and neuropeptides in the cutaneous tissues as structures for the biomolecules mediated axon reflexes. Recent advances have determined that NO and calcitonin gene related peptide play crucial roles in the comprehension of the axon reflex. The stimuli-evoked axon reflex and NOergic biomolecules/neuropeptides increase local blood flow with higher levels in acupoints/meridians, which move radioactive substances or tracer dyes in the skin and subcutaneous tissue under a linear path resembling acupoints and meridians, the important phenomena of meridians induced by the stimuli. The evidence and understanding of the biomolecular processes of the tracers along a linear pathways resembling meridians have been summarized with an emphasis on recent developments of NO and neuropeptides mediating stimuli-evoked axon reflexes to increase local blood flow with higher levels in acupoints/meridians, which move radioactive substances or tracer dyes in the skin and subcutaneous tissue contributing to tracers along linear pathways resembling meridians in this mini-review.

INTRODUCTION

The Meridian System (channels and collaterals, jingluo) according the description of Traditional Chinese medicine (TCM) is an essential pathway system that deal with physiological regulation and reflects pathological changes of the human body. Acupuncture points (acupoints) are located along the 12 main meridian pathways, and stimulation and/or activation of acupoints/meridians have been used in many TCM practices such as acupuncture, electrical acupuncture (EA), transcutaneous electrical nerve stimulation (TENS), moxibustion (heating acupoints), Qi Gong and meditation [1-3]. It is described that the meridian systems flow “meridian qi”, which serves as energy exchange, movement, and connection from the surface of the body to the internal organs, as well as communication with the universe, environment and our body as a whole. Various diseases are associated with blocked meridians, and acupuncture stimulates acupoints on the body, releasing and easing the “meridian qi” for treating disorders. There has been a widespread and increasing interest in scientific examinations exploring the Meridian System and the use of acupuncture for treatment of disorders over the world. However, the most logical and direct approach to identify a specialized anatomic entity of acupoints and meridians are still lack [1-4]. The chemicals, structure, functions and mechanisms of the meridians are unclear. One of the 125 major exploration and discovery questions is that “Is there a scientific basis to the Meridian System in traditional Chinese medicine?” as the international frontier, global common needs and gathering foresight released in 2021 by Shanghai Jiao Tong University and "Science" magazine [4].

Over the last few decades there has been an increasing evidence from biophysical, physiological, and biochemical approaches implicates some of specificity for the acupoints in the body around the world. There are various studies from biophysical approaches dealing with specificity for the acupoints and/or median lines in the body [2,3,5,6]. Although no well-defined anatomic entity is available, an abundance of information has now accumulated several meridian phenomena. It is generally accepted that the most commonly observed and internationally identified meridian phenomena are: 1) the characteristics of low electrical resistance and high electric conductance of acupoints/meridians; 2) the propagated sensation (numbness, pressure, heaviness, warmth, and/or radiating paranesthesia from the subjects) along meridian pathways (PSCP) induced by local stimulations of distal acupoints in the same meridian; and 3) radioactive or fluorescent tracers along a linear path resembling acupoints and meridians [2,3,5-10], as shown in figure, right panels.

Figure.

Representation of NOergic molecules, neuropeptides, and other biomolecules mediated stimuli-evoked peripheral axon reflexes involved in tracers along a linear pathways resembling meridians is illustrated in the left top panel, morphological studies of skin tissues with acupuncture points (Acupoints)/meridian are shown in the left bottom panel, and the visualized the fluorescent line along the forearm following injection of the dye into acupoint PC6 is shown in the right panel.

The left top panel is a schematic model of NOergic molecules, neuropeptides, and other biomolecules mediated stimuli-evoked peripheral axon reflex, which is elicited by a stimulus such as acupuncture, transcutaneous electrical nerve stimulation, and electrical heat applied on the skin acupoints of the arms or legs. All experiments of the tracers along a linear pathways resembling meridians have been conducted by injection of a radiotracer solution or tracer dyes in a volume of solution into acupoints. Solution injected into acupoints produce much stronger mechanical stimuli than acupuncture stimulation, which cause axon reflex.

The left bottom panel show acupoints/meridians exist higher number of nerve fibers/trunks, blood vessels, hair follicles, and sweat glands demonstrated by anatomical studies. The higher number of small nerve fibers, blood vessels in acupoints/meridians with rich endothelia and neuronal NO synthase, TRPV1 and neuropeptides such as calcitonin gene related peptide (CGRP), and sweat glands in the cutaneous tissues serve as structures for NO signaling molecules and neuropeptides mediated axon reflexes with signal functions. After injection of a radiotracer solution or tracer dyes in the solution into an acupoint, NOergic biomolecules and neuropeptides are increased to evoke axon reflex one after another by the mechanical stimuli, which increase local microcirculation and blood flow with higher levels in acupoints/meridians, then, move radioactive substances or tracer dyes in the skin and subcutaneous tissue under a linear path resembling acupoints and meridians.

The right panels show a fluorescent line 24 hours after injection 0.1 ml of fluorescein sodium into PC6, which induced mechanical stimuli (a), and the fluorescent line was showed on the forearm along the PC meridian line following injection of the dye into the acupoint; the visualized tracer pathway along the forearm is compared with the PC meridian line marked acupoints PC5, PC4, and PC3 following acupuncture map before injection (b) (black dots). These PC meridian line/acupoints from acupuncture map and traced line induced by the injection closely approximated each other (from Li TJ et al.: In-vivo visualization of pericardium meridian with fluorescent dyes. Evid Based Complement Alternat Med. 2021; 2021: 5581227.).

Acupoints are located along meridian lines on the body surface, described in TCM [2,5] while whether-or-not acupuncture points “exist” has been given too little attention within the acupuncture research community [11]. Anatomical studies have identified that the number of nerve fibers/trunks, blood vessels, hair follicles, and sweat glands are enhanced over acupoints compared to their adjacent control areas [1,12,13], as shown in figure, left bottom panel. Recent review summarized that L-arginine-derived nitric oxide (NO) in the skin contributes to electrical conductance of acupoints through the increases in NO release, which enhances norepinephrine (NE) production and activates transient receptor potential vanilloid type-1 (TRPV1)] as well as increases release of neuropeptides, and these signaling molecules involved in the skin low resistance at acupoints [3,14-17]. Animal studies showed that NO level is elevated by EA in the acupoints/meridians in rats associated with an enhanced expression of NO synthase endowed with TRPV1[18-20]. Dermal microdialysis in humans reported that NO-cGMP releases are increased by electroacupuncture (EA) in the subcutaneous tissue of acupoints [21]. Several studies demonstrated that transcutaneous electrical nerve stimulation (TENS), manual acupuncture (MA), and electrical heat produce an elevation of NO release predominantly over acupoints [22-24]. Consistently, these stimuli, TENS, electrical heat, and EA have been used to elicit axonal reflexes, which are characterized by local release of NO and calcitonin gene related peptide mediated by activation of TRPV1 [25-30]. NO signaling molecules and neuropeptides [calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY)] involve in the stimuli-evoked axon reflexes [28-30], and the same stimuli-elicited biomolecules-mediated axon reflex may also participate in the somatosensory signal transduction and processes of the PSCP, which have been identified over the body surface along the meridian lines in certain aspects of the subject's perceptions (sensory, affective and cognitive), including numbness, pressure, heaviness, warmth, and/or radiating paranesthesia [31,32]. In this paper, the evidence and understanding of the biophysical approaches of acupoints/meridians have been summarized with an emphasis on recent development of stimuli-evoked NO release through the axon reflex and neuropeptides mediating meridian phenomenon, particularly tracers along a linear pathway over the body surface which are similar with the acupoints and meridian lines described in the map of acupoints and meridians.

Studies of tracers along a linear path resembling acupoints and meridians

Several groups of the investigators have identified the slow-migrating pathways of radioactive tracers along a linear pathway over the body surface similar with the map of acupoints and meridian lines [9,33-38]. Early studies [39] reported that meridians can be made visible by the shape of radioactive tracer pathway following injection of a radiotracer solution into an acupoit. Migrating pathways of radioactive tracers following injections of isotope (9mTc 300-500 μci, about 30-70 μl solution) into acupoints are in straight correspondence with classical course of meridians but are quite different of lymphathic vessels in patients and healthy subjects [40]. The appearance of radioactive paths have been also confirmed after the injection of a radiotracer (Technetium 99m in a volume of 0.05 ml) at acupoints in healthy humans by Lazorthes et al. [41]. A linear radioactive tracer over the skin surface, which appeared and traveled at approximately 2.5 cm/minute for a length of 11 cm hypodermically from the injected site, was induced by injection of (99m Tc in a volume of 0.125 ml) sodium pertechnetate but not produced by other three nuclear tracers, 201Tl-thallous chloride in a volume of 0.3 ml, (131I) iodine-sodium in a volume of 0.05 ml, and (99mTc in a volume of 0.125 ml) rhenium sulfide injected into acupoints identified by measurement with low-electrical resistance on the legs of adult dogs [33]. Direct injections of the dyes into blood vessels induced a much more instantaneous appearance of the radioactive tracer over the skin surface following a subsequent disappearance of the trajectory in 10 seconds. However, the radioactive tracer was detected at axillary lymph nodes at 90 minutes after injection of (99mTc) rhenium sulfate in both acupoints and controls, and the compound has a predilection for lymphatics but did not have any linear pathway over the skin. For the reasons, the authors concluded that veins or lymphatic vessels could not have been responsible for the linear radioactive tracer over the skin surface following (99m Tc) sodium pertechnetate injections [9,33,34]. Linear and rapid migration of 99mTc-pertechnetate was not altered or prevented by incisions that did not intersect the migration pathway, but was abolished by the incision which was filled with petroleum jelly, or with a solid silicone sheet [35]. Therefore, these investigators concluded that capillary electrophoresis is suggested to explain the hypodermic diffusion of inert particles through specific and constant linear pathways [9,33-35].

In addition, previous studies showed that fluorescein injected into acupoints produced similar visualization of tracers along a linear pathway with low hydraulic resistance in the body along the meridians in rats and mini-pigs [42-44]. Several studies reported that injection of tracer dyes at acupoints led to linear migration of the dye along a linear pathway similar with the meridian map as described [42-45]. Low hydraulic resistance channels were found very close to low impedance points along meridians, which were imaged through injection of Alcian blue and isotopic tracing [42,43]. Animal studies reported that visualization of interstitial channels along meridians with average length of the migration tracks was 5.13 cm following injections of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of sodium fluorescein into acupoints in mini pigs [44]. Similar results were also observed in rats [46]. Some studies have shown fluid flow along venous adventitia in rabbits [45], “perivascular-like-spaces” along the meridians histologically [47], and low hydraulic resistance channels along conception vessel in rats [46], which suggest that fluid in blood vessels are involved in tracers along a linear path resembling acupoints and meridians. However, other experiments have identified that the tracers along a linear path resembling acupoints and meridians are not directly attributable to lymphatics or blood vessels [9,33,34]. Following injecting a small amount of paramagnetic contrast or gadopentetate acid dimeglumine into acupoints, subcutaneous pathways have been visualized in humans, which have a characteristic “puncture-resistant” appearance, indicating a non-tubular structure, not consistent with lymphatic or blood vessels [48]. Fluorescein sodium is found in the pathways of hind limbs and segments of the small intestines after injecting 0.2~1 mL of fluorescein sodium in acupoint (KI3) of healthy rabbits [45]. Consistently, recent research reported that injection of 0.1 mL of fluorescent dyes into the acupoint PC6 at the forearm, 15 (79%) of fluorescein sodium injection (but not indocyanine green) in the subjects were associated with slow diffusion of the dye proximally along a linear pathway over the forearm matching closely with the Pericardium meridian as described in the meridian map in humans (Figure, the right panels) [49]. The observed migratory speed, path trajectory, and selective association with Technetium-99 (99mTcO4, but not other nuclear dyes) led authors to speculate that paths were attributed to a yet undescribed channel with low hydraulic resistance through the extracellular matrix (42-44,47), and not to vein or lymphatics [9,33,34].

Stimuli-evoked NOergic molecules and neuropeptides mediated axon reflexes contribute to tracers along a linear pathway over the body surface which are similar with the meridian lines described in acupoints and meridian map

In order to examine the radioactive or fluorescent tracer pathway along a linear pathway over the body surface similar with described the map of acupoints and meridians, all experiments have been conducted by injection of a radiotracer solution or tracer (dyes) in a volume of either 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 or 1.0 mL into an acupoit [9,33-35, 44,49]. Needle injection is a mechanical stimulus and the injected solution is an additional extension stimulus, both of them produce stimuli-evoked axonal reflexes [50,51]. Acupoint injection of solution or drugs has a better effect than traditional acupuncture achieving the purpose of treatment by much stronger stimulating the acupoints [52-54]. Intra-dermal injection of hypertonic saline produces an axon reflex flare response involved neural and vascular mechanisms, and the axon reflex vasodilatation measured by a blood flow value which is inhibited by lignocaine comparably to its effects on histamine and substance P [50,51]. Axonal reflexes have been defined as a response elicited by mechanical, electrical, thermal, or pharmacological stimuli [55,56], and are characterized by local release of NO [30] and neuropeptides (CGRP mediated by activation of TRPV1 and NPY), as shown in figure, in the left top panel [26-29]. Moreover, studies have shown that local NO release is consistently induced by MA, EA, TENS, and electrical heat [22-24]. These stimuli, MA, EA, TENS, and electrical heat, have been demonstrated to evoke axon reflexes in various studies [55,56]. Peripheral axon reflexes play an essential role in the regulation of skin blood flow, and activation of axon reflexes cause vasodilatation and increase local blood flow (26,28-30). It appears that injection of a radiotracer solution or tracer dyes in a volume of solution cause much stronger stimuli than acupuncture stimulation at local acupoints, which produced stimuli-evoked axon reflexes and elevations of NO and neuropeptides levels. The stimuli-evoked axon reflexes and NO signaling molecules/neuropeptides allows for a flare of blood flow, leading to moving radioactive substances or tracer dyes in the skin and subcutaneous tissue under axon reflex pathways (Figure, the left top panel). Therefore, the radioactive or fluorescent tracer pathway along a linear path represent a flare of blood flow following stimuli-evoked axon reflexes.

Regarding the radioactive or fluorescent tracer pathway along a linear path resembling acupoints and meridians, it has been concluded “As for the meridians themselves, in 1987, scientists who analyzed the movement of radioisotopes in the body concluded that meridians are actually spaces within the body, not structures. More recently, a 2020 publication in the journal research reached the same conclusion [4]” The mechanisms of the linear and rapid migration of 99mTc-pertechnetate have been examined by various methods including local applications of skin incisions, filled with petroleum jelly, or with a solid silicone sheet [35], the authors concluded that veins or lymphatic vessels may not be the sources responsible for linear and rapid migration of 99mTc-pertechnetate over the body surface similar with the acupoints and meridian lines [9,33,34]. The conclusion is consistent with recent study using injection of fluorescent dye tracer and observation by both ultrasound imaging and vein locating device in humans, which showed the visualized tracer pathway along the forearm following injection of the dye into acupoint correlated to the distribution of intermuscular fascia in the areas but did not correlate to any corresponding vessels (arterial or venous) or lymphatics in the locations (Figure right panels) [49]. Kovacs et al. (2000) indicated that capillary electrophoresis is suggested to explain the hypodermic diffusion of inert particles through specific and constant linear pathways [34]. Our analyses agree with this suggestion and further point out that the stimuli-evoked axon reflex and NO signaling molecules/neuropeptides produce a flare of blood flow, leading to moving radioactive substances or tracer dyes in the skin and subcutaneous tissue under a linear path resembling acupoints and meridians. NO level and NO synthase expression are consistently increased in the subcutaneous tissue of acupoints and meridian regions compared to non-meridian areas in rats [57] associated with an enhanced expression of NO synthase endowed with TRPV1 [18,19]. Our previous studies have demonstrated that NOS protein levels and NO contents are consistently higher in skin regions containing acupoints/meridian lines compared to control tissues in rats [21,57]. Acupuncture and transcutaneous electrical nerve stimulation increased expressions of endothelial and neuronal NO synthases but not expression of inducible NO synthase [57-59]. It has been demonstrated that NADPH diaphorase positive neurons are distributed in the ST36 point and some of the neurons are projected from the lamina IX of L4 to S1 in spinal cord [60]. These results [57-60] suggest that the stimuli-evoked NO release are endothelial and neuronal NO which do not cause toxicological effects. Several studies have demonstrated that MA, EA, TENS, and electrical heat-induced elevations of NO release over skin regions with a high level at acupoints [22-24]. Axon reflexes-mediated NO and CGRP releases are also induced by the same stimuli [27-30] and acupoint injection of solution [50,51]. Cutaneous small nerve fibers encompass unmyelinated C-fibers and thinly myelinated Aδ-fibers, which innervate dermal vessels (vasomotor fibers), sweat glands (sudomotor fibers), and hair follicles (pilomotor fibers) participate axon reflex [26,55,56]. Quantitative assessment of distal autonomic innervation based on axon-reflexes for assessment of peripheral neuropathies and Parkinson's disease-related pathology [61,62]. Anatomical studies have demonstrated that acupoints/meridians exist higher number of nerve fibers/trunks, blood vessels, hair follicles, and sweat glands [1.12,13]. It appears that the higher number of small nerve fibers and blood vessels with rich NO synthase and neuropeptides, and sweat glands contribute to higher NO related molecules/neuropeptides and axon reflex-evoked blood flow with higher levels in acupoints/meridians, leading to moving radioactive substances or tracer dyes in the skin and subcutaneous tissue under a linear path resembling acupoints and meridians. Therefore, this review suggests that the higher number of small nerve fibers and blood vessels with rich NO synthase, NE, TRPV1, and neuropeptides, and sweat glands in the acupoints/meridians serve as structures, which contribute to biophysical characteristics of low electrical resistance and tracers along a linear pathways resembling meridians.

Conclusion

Over the last few decades, there are various studies from biophysical approaches dealing the phenomena of meridians. An abundance of studies from different international groups have demonstrated that acupoints possess characteristics of low electrical resistance and high electric conductance, the PSCP, and tracers along a linear pathways resembling meridians are the most commonly observed and identified meridian phenomenon. In many texts, although the meridian phenomenon to be genuine evidences for the functional existence of acupoints/meridians, there is a lack of objective and systematic experimental studies to support biochemical and morphological basis of the meridians. It is well known that the actual value of skin electrical resistance depends upon the activity of the sympathetic nervous system and that blocker of sympathetic function enhances the skin resistance [63,64]. A recent review summarized that NO concentrations are enhanced in skin acupoints/meridians, and L-arginine-derived NO synthesis and noradrenergic transmission modify skin electric conductance, which contributes to low resistance characteristics of acupoints and meridians [3]. 3H-NE synthesis/release was enhanced in acupoints and facilitated by presence of an exogenous NO donor and inhibited by an inhibitor of NO synthesis [6]. NOergic signaling molecules (NO-NE/TRPV1) and neuropeptides play important roles in mediating the skin conductance responses to electrical stimulation and contribute to low electrical resistance and high electric conductance of acupoints/meridians. Animal studies showed that NO level is elevated by EA in the acupoints/meridians in rats associated with an enhanced expression of NO synthase endowed with TRPV1 [18,19]. Dermal microdialysis in humans reported that NO-cGMP releases are increased by EA in the subcutaneous tissue of acupoints. Several studies demonstrated that TENS, MA, and electrical heat produce an elevation of NO release predominantly over acupoints. Consistently, these stimuli, TENS, electrical heat, and EA have been used to elicit axonal reflexes, which are characterized by local release of NO and neuropeptides mediated by activation of TRPV1. NO signaling molecules and neuropeptides (CGRP, SP, and NPY) involve in the stimuli-evoked axon reflexes, and the same driver elicited biomolecules-mediated axon reflex may also participate in the processes of PSCP and tracers along a linear pathways resembling meridians.

Moreover, all experiments of the tracers along a linear pathways resembling meridians have been conducted by injection of a radiotracer solution or tracer dyes in a volume of solution into acupoints. Solution injected into acupoints produce much stronger mechanical stimuli, which cause axon reflex. Although more sophisticated approaches are requested to address biochemical-physiological interactions in the processes in this fields, the results from animal and human studies consistently suggest that the stimuli-evoked axon reflex and NOergic biomolecules/neuropeptides increase local blood flow with higher levels in acupoits/meridians, which move radioactive substances or tracer dyes in the skin and subcutaneous tissue under a linear path resembling acupoints and meridians, the important phenomena of meridians induced by the stimuli. Anatomical studies have demonstrated that acupoints/meridians exist higher number of nerve fibers/trunks, blood vessels, hair follicles, and sweat glands. The higher number of small nerve fibers, blood vessels with rich endothelia and neuronal NO synthase, TRPV1 and neuropeptides, and sweat glands in the cutaneous tissues serve as structures for NO signaling molecules and neuropeptides mediated axon reflexes resulting in low electrical resistance, PSCP, and tracers along a linear pathways resembling meridians. The increased interest in the meridian phenomenon has led to an open-minded attitude towards understanding this system, which is fundamental important to establish the valid aspects of scientific basis of Chinese medicine mechanisms and related therapies.