Consistent with the physiological characteristics of acupoints7,8,9,13, our study showed that neurogenic spots caused by activation of somatic afferents during visceral disorders: (1) are found most frequently in the same anatomical location as traditional acupoints, (2) reveal high electrical conductance, and (3) show mechanical hypersensitivity. The linkage of neurogenic spots on the skin to internal organs was verified in a hypertensive rat model by showing that several sensory neurons exhibit branching to both the skin and the heart. Lastly, stimulation of neurogenic spots produces acupuncture effects mediated by the endogenous opioid system. The present study suggests that acupoints associated with internal organs may be identical to neurogenic inflammatory spots occurring on the skin that are associated with visceral disorders.

Neurogenic spots anatomically correspond to traditional acupuncture points

Noxious signals from viscera frequently produce referred pain at somatotopically distinct body surfaces, which is generally attributed to viscerosomatic convergence at the spinal cord segments15. In the somatic area of referred pain, neurogenic inflammatory spots are found and can be easily visualized by intravenously injecting Evans blue dye17. In the present study, most of the neurogenic spots in the hypertensive rats were found in the dermatome, which is innervated by the same spinal segments (C8-T2) that innervate the heart22, and 67% of those spots matched with acupoints, such as PC6, PC7, and HT7. The acupoints are prescribed most frequently for cardiac disorders1 or have been shown to be effective in cardiovascular disorders. In support of these findings, multiple studies have shown that acupuncture stimulation at PC6 can improve or treat cardiovascular disorders, including hypertension, myocardial infarction or hypotension, in humans or experimental animals23,24. In contrast, colitis rats revealed neurogenic spots mainly in the hind paw and frequently in the lower back, thighs or tail, which is consistent with our previous study25. The spots were found in the dermatome corresponding to the spinal cord sections L2-S2, as mapped by electrical stimulation of C-fibres in the spinal nerve in rats26. Given that the nerves innervating the colon project to the T13-L2 and L6-S1 spinal cord segments and those of hind paw primarily enter at the L4-L5 spinal segments, there seems to be no overlapping spinal segments between the colon and hind paw. However, the spinal neurons in nearby segments, including L4-L5, are known to be activated during colon inflammation27, providing the neuroanatomical basis for the occurrence of the neurogenic spots in the hind paw in the colitis rats. The spots activated by colitis also corresponded to acupoints such as SP4, ST44, and BL66, which are points commonly used in acupuncture clinics for the treatment of gastrointestinal disorders1. These results indicate that many neurogenic spots are found in the dermatome of segmentally related organs and correspond to the location of acupoints commonly used in acupuncture medicine for the treatment of visceral disorders. It is noted that the number and distribution of neurogenic spots in the skin varied among rats with hypertension or colitis (Fig. 1 and Supplementary Table S1). In line with our findings showing DRGs double-labelled with DiI and FG (Fig. 5B), DRG neurons with dichotomized afferents innervating both skin and viscera have been reported28 and are considered as a mechanism of referred visceral pain17. However, the degree of dichotomized afferents varies considerably among subjects29. This variability may be linked to the variations in neurogenic spots among rats in this study.

Stimulation of neurogenic spots produces acupuncture-like effects via the endogenous opioid system

An important characteristic of acupoints is that their stimulation should produce therapeutic effects on the associated visceral disorders1,2. The present study showed that acupuncture at neurogenic spots attenuated the development of hypertension or colitis. However, these effects were not reproduced by acupuncture stimulation of either a nearby site 3–5 mm away from the neurogenic spots or a non-neurogenic acupoint, PC4, which is used in acupuncture clinics for the treatment of hypertension1. These results suggest that when stimulated with acupuncture, neurogenic spots generate therapeutic effects on the associated visceral disorders and that neurogenic spots may represent truly effective acupoints rather than conventional acupoints, which is supported by our previous study showing that GV1, an acupoint between the tail base and the anus, reveals neurogenic inflammation in colitis and has therapeutic effects on colitis when acupuncture is applied25. Our data are consistent with previous animal studies showing that needling at sham or inactive acupoints, located 3–5 mm from active (verum) acupoints, is ineffective or less effective than needling at active acupoints30,31. It is known that sensory nerve endings are distributed unevenly over the body and acupoints have a higher density of sensory nerve endings than surrounding areas32. Moreover, previous studies have suggested that active acupoints are associated with tissues where the sensory nerve endings are sensitized by neurogenic inflammatory mediators33,34. Given that the sensitized sensory nerve endings are more sensitive to external stimuli than intact sensory nerves, active acupoints with a high density of sensory nerve endings are sensitized by neurogenic inflammatory mediators under pathological conditions. Therefore, stimulation of these sensitive points would generate therapeutic effects by reaching physiological thresholds, compared with stimulation of normal surrounding tissues, such as sham or inactive acupoints. However, in systemic reviews of clinical trials, stimulation of sham acupoints can elicit similar effects as stimulation of verum acupoints in human subjects35. Conversely, experimental animal studies have consistently reported the specific effects of active acupuncture are not produced by stimulation of sham acupoints in awake or anesthetized rats30,31. The positive effects of sham acupoints in humans may be associated with placebo responses36 or inadequate selection of sham acupoints that are several millimeters apart from active acupoints but still located in the effective zone of active acupoints. If the present strategy can be applied to identify active acupoints in human subjects, it may help assess the validity of acupoint specificity by differentiating active acupoints from inactive (sham) acupoints.

It is known that stimulation of small diameter (C and Aδ) nerve fibres during acupuncture treatment generates a sensation (called “Deqi”) and acupuncture effects9,13. The major part of the “Deqi” sensations, which manifest as pressing, numbing, dull, cold and throbbing sensations, is elicited by small fibre stimulation in acupoints. As shown by enhanced CGRP expression (Fig. 4C), the neurogenic spots display small afferent fibre activation17, which may evoke or mediate the acupuncture-specific “Deqi” sensations during needling. Furthermore, the present study showed that small fibre activation by injecting capsaicin or mustard oil into neurogenic spots attenuated the development of hypertension, suggesting that the effects of neurogenic spots are generated through the activation of small diameter nerve fibres. Similarly, a previous study revealed that traditional or electrical acupuncture applied to the PC5-6 acupoints activate small fibres to evoke cardiovascular effects37. Taken together, these findings suggest that neurogenic spots may require the activation of small afferent fibres to produce therapeutic effects in a similar manner as traditional acupuncture.

Cumulative evidence suggests that various effects of acupoint stimulation are mediated through the endogenous opioid system11,38,39. Stimulation of effective acupoints exerts strong influences on endogenous opioids in the brain and the released opioids play critical roles in producing various acupuncture effects. The endogenous opioid system is considered a main pathway for the onset of various acupuncture effects40. A previous study showed that acupoint stimulation activates opioid receptors in the RVLM, a central site for the regulation of blood pressure41, and suppresses visceral reflex stimulation-induced hypertension42. Similarly, in the present study, the anti-hypertensive effects of neurogenic spot stimulation were abolished by pretreatment of the RVLM with naloxone and replicated by the administration of morphine. Our previous study revealed that acupuncture at the neurogenic GV1 point alleviates diarrhea and colonic inflammation, indicated by enhanced MPO levels, via the endogenous opioid system, and suggested that the increased opioid peptides would generate anti-inflammatory effects by decreasing the enhanced levels of MPO and pro-inflammatory cytokines in the colon43,44. These findings suggest that the effect of the stimulation of a neurogenic spot recruits the endogenous opioid system in a similar manner as acupoint stimulation.

Neurogenic spots reveal the same physiological features as acupuncture points

Acupoints are frequently described as having electrically distinct properties, including high electrical conductance and potential, low impedance and resistance, and increased capacitance8,9. The physiological mechanisms underlying these electrical properties in acupoints have not been elucidated. In the present study, the electrical conductance at neurogenic spots was increased with the development of hypertension and higher in neurogenic spots than in the nearby skin, suggesting that neurogenic spots share electrical properties with acupoints. The present study also showed increased CGRP expression, markedly microvessel dilation in the dermis of neurogenic spots and plasma extravasation (as detected by Evans blue dye). CGRP released by activation of small fibre sensory nerve terminals causes neurogenic inflammation in the skin by activating vasodilation, axon reflex flare, and microvascular plasma extravasation18,45. CGRP has been reported to be involved in the peripheral mechanism of acupuncture effects46. Thus, we suggest that the vasodilation and plasma extravasation by local tissue release of CGRP increases skin moisturization and the sub-skin tissue water content, increasing electrical conductance at neurogenic spots, which may underlie the electrical properties in acupoints.

It is generally accepted that acupoints become sensitive under pathophysiological conditions of internal organs5,6,7. Chae, et al.6 reported the increased sensitivity of the acupoint SP6, which is clinically used for reproductive disorders1 in women undergoing premenstrual syndrome. In a clinical study, patients with gastric ulcers or gastritis showed significantly decreased pressure-pain thresholds at the disease-related acupoints compared to healthy subjects5. In the present study using the von Frey method, the mechanical thresholds of neurogenic spots were decreased with the development of hypertension and colitis, indicating the increased mechanical sensitivity of neurogenic spots. Because the neuropeptides CGRP and SP are released during neurogenic inflammation and induce mechanical and thermal hyperalgesia in the skin47,48, the increased sensitivity of neurogenic spots may be attributed to enhanced release of CGRP, as observed in the CGRP immunohistochemistry results. This result suggests that tenderness (an increase in sensitivity) of neurogenic spots under pathological conditions overlaps with the characteristic of traditional acupoints.

TCM describes that each acupoint is linked to a specific internal organ, particularly the acupoints lying along the Heart (HT) or Pericardium (PC) meridians (i.e., PC6, PC7 and HT7) on the medial aspect of the arm, which are internally connected with the heart1. The linkage of neurogenic spots on the skin to internal organs was identified by our retrograde study showing convergent DRG neurons innervating both the heart and the neurogenic spots. This finding is in agreement with that of a previous study showing that 7–14% of DRGs from the cardiac area labelled with true blue are simultaneously are labelled with nuclear yellow injected into the ulna nerve in rats28. The connection between acupoints and the internal organs in TCM may be explained by the viscero-somatic convergence that occurs at neurogenic spots.

In conclusion, the present study suggests that traditional acupoints associated with internal organs represent one form of neurogenic inflammation occurring on the skin that is associated with visceral disorders.