Neuropeptide Initiated Mast Cell Activation by Transcutaneous Electrical Acupoint Stimulation of Acupoint LI4 in Rats

Transcutaneous electrical acupoint stimulation (TEAS) has been consistently used clinically for its ease of operation, non-invasiveness and painlessness, in contrast to the characteristics of inserted needles. However, the mechanism remains unknown. The aim of this study was to investigate the local response of TEAS at Hegu acupoint (LI4). Immunohistochemistry was used to measure the expression of tryptase-positive mast cells, neuropeptides of the calcitonin gene-related peptide (CGRP) and substance P (SP) in LI4. Mast cells were also labelled with serotonin (5-HT), neurokinin-1 receptor (NK-1R) and toluidine blue. The results showed that cutaneous CGRP and SP immune-positive (CGRP-IP or SP-IP) nerve fibres in LI4 were more highly expressed. There were high degrees of mast cell aggregation and degranulation with release of 5-HT near the CGRP-IP or SP-IP nerve fibres and blood vessels after TEAS. The degranulation of mast cells (MCs) was accompanied by expression of NK-1R after TEAS. Either mast cell membrane stabilizer (Disodium cromoglycate) or NK-1R antagonist (RP 67580) diminished the accumulation and degranulation of MCs induced by TEAS. Taken together, the findings demonstrated that TEAS induced sensory nerve fibres to express CGRP and SP, which then bound to the NK-1R on MCs, after which MCs degranulated and released 5-HT, resulting in TEAS-initiated acupuncture-like signals.

Acupuncture is a medical technique that involves insertion of needles into acupoints to relieve pain and regulate functions of internal organs. Because of its curative effects and few side effects, acupuncture has been practiced in more than 183 countries and areas. Studies showed that the therapeutic effects of acupuncture were mediated by nervous system 1-3 , immune system 4 and other systems.
There are several manipulation methods used to stimulate acupoints, including acupressure, manual acupuncture (MA) and electroacupuncture (EA). Although MA is widely used for its ease of manipulation and rapid efficiency, it depends highly on the skill of the acupuncturist and is difficult to duplicate. EA became popular for its adjustable strength, frequency and easy quantification in the clinic. However, some people are afraid of the painful sensation of puncturing, and the puncture method is highly dependent on the doctor's technique.
Transcutaneous electrical acupoint stimulation (TEAS) combines the transcutaneous electrical stimulation of physical therapy with acupoint therapy but less invasive than MA 5 . Increasing evidence supports the notion that TEAS is effective in reducing pain during labour 6 , treating reproductive disorders 7 , preventing and treating nausea and vomiting in patients receiving electroconvulsive therapy 8 , and improving the pregnancy rate and implantation rate in patients with implantation failure 9 . Transcutaneous electrical nerve stimulation (TENS) or TEAS instead of MA or EA is becoming a trend for they had same effect on antinociception and shares common neural mechanisms 10 . Our previous study demonstrated that local cutaneous nerve terminals and mast cells (MCs) responded to MA. MA at acupoint LI4 11 induces higher expression of neuropeptides of calcitonin gene-related peptide (CGRP) and substance P (SP) in subepidermal nerve fibres to activate MCs. Whether nerve-mast cell cross-talk contributes to the effects of TEAS remains unclear. The present study focused on local histologic and Sciences, China Academy of Medical Sciences (experimental animal license number: SCXK(Jing)2014-0013). Rats were housed in standard animal facilities in which the room temperature was maintained at 24 ± 2 °C, the humidity was 60-70%, and the noise levels were lower than 60 dB. All animals were grouped in twos or threes with ad libitum access to food and water. The bedding material and drinking water were replaced every day to keep the cages clean and dry. The animals were maintained on a standard 12-hour light-dark cycle (dark cycle 8:00 PM-8:00 AM) and were allowed to acclimate to the housing conditions for seven days prior to the experiment.
The experiment was performed on 15 adult male Sprague-Dawley rats with weights of 180-220 g, Under anaesthesia with 10% urethane (1 g/kg), TEAS was applied at the right Hegu acupoint (LI4), which is located on the radial side at the midpoint of the second metacarpal bone, as shown in Fig. 1. The parameters applied were a frequency of 2/100 Hz and a current of 10 mA to cause obvious shaking of the front paws for 20 min, consistent with those parameters used in routine clinical treatment or basic research 6,11 . The contralateral LI4 of the same rat was used as the non-stimulated control.
After an initial wash in 0.1 M PBS (pH 7.4), the tissues were preincubated in a solution of 3% normal goat or donkey serum and 0.5% Triton X-100 in 0.1 M phosphate-buffered solution (PB, pH 7.4) for 30 min to block non-specific binding. The sections were then incubated with primary antibodies for 24 hours at 4 °C. After washing in 0.1 M PB for 3-10 min, goat anti-mouse Alexa Fluor 488 or 594 secondary antibody (1:1000, Molecular    Toluidine blue staining. The sections were also stained by toluidine blue to label MCs. The sections were first stained with 0.1% toluidine blue (formulated with distilled water) for approximately 15 seconds and then quickly washed with distilled water three times to remove floating colour. Next, 30% alcohol (formulated with PB) was used to separate colours, and sections were dehydrated by sequentially dipping them quickly (approximately 5 seconds) in 75%, 85%, 95% and 100% alcohol. Finally, sections were dipped in xylene for 5 minutes and mounted with neutral resin sheet. After staining, randomly selected sections were examined under light microscopy. An intact MC showed deep blue staining; while degranulated MC showed purple or red staining with granules extruded adjacent to the MC. MC degranulation was determined as described previously 15 .

Discussion
In this study, we showed that TEAS induced high expression of CGRP-IP and SP-IP nerve fibres to activate MCs by NK-1R. The activated MCs aggregated with degranulation of 5-HT near the CGRP-IP, SP-IP sensory nerve fibres and blood vessels. This response conveyed TEAS signals to certain pathways.
TEAS is a handy therapy for patients to continue treatment at home. The mechanism of TEAS is important for its promotion. Previous studies showed that needling reactions included neuronal, biophysical and biochemical components. All types of somatic afferent nerve fibres (I-IV) were activated by MA stimulation, eliciting various effects 3,18 . MC activation at acupoints was important for the EA effect against pituitrin-induced bradycardia in rabbits 19 . The degranulation of MCs participated in the effects of acupuncture 19,20 , moxibustion 21 , and laser acupuncture 22 . Our previous study 11 showed that MA stimulation induced high expression of nociceptive neuropeptides of SP and CGRP in subepidermal nerve fibres to activate MCs. MCs aggregated and degranulated, releasing tryptase, 5-HT and HA. These neuroactive components played a key role in conveying acupuncture signals to various pathways to deliver the effects of acupuncture. Present data provided evidence that TEAS also induced MCs aggregation and degranulation with release of 5-HT around the sensory nerve fibres and blood vessels (Fig. 10), suggesting that TEAS and MA induced the same dermal and sub-dermal changes at the same acupoint (LI4).
Neuropeptide receptors NK-1R and NK-2R were found to be largely localized on MCs, and the increased expression of NK-1R and NK-2R on MCs played a role in the MC-nerve association 23 . SP was a neurotransmitter in relation to MCs activation by NK-1R 17 and this action can be diminished by application of NK-1R antagonist 24 . NK-1R antagonists bind to NK-1R on the MC, resulting in competitive or noncompetitive inhibition of the SP/ NK-1R signalling pathway. In the present study, we observed that expression of NK-1R increased in MCs and MCs were activated by TEAS. The activation was diminished by NK-1R antagonist or mast cell membrane stabilizer. It indicated that SP receptor NK-1 on MCs played an important role in local response at LI4 of TEAS, which strongly supported the hypothesis.
TEAS is "acupuncture-like TENS" and ideally combined acupoint and bioelectricity. It should be noted that electrical stimulation can also modulate MCs secretion directly. Electrical stimulation of the cholinergic hypoglossal nerve caused a progressive degranulation and there was a close anatomical association between MCs and pre-terminal axons. The distance between the plasma membranes of the MCs and pre-terminal axons was less than 100 nm and in some points they seemed to be in contact 25 . Therefore, not only the cholinergic nerve but also sensory nerve by electrical stimulation activates the MCs, which indicates that activation of the nerve fibres is important in initiating the local response of TEAS. Recent studies proposed that a mechano-transduction pathway participated in MC-nerve cell interactions at the acupoint. MCs also transduced mechanical stimuli to acupuncture signals by activating histamine H1 and adenosine A1 receptors 26 through TRPV2 channels. This results suggested that the MC-nerve functional unit was a key component of physiologic and pathophysiologic responses 27 , especially after TEAS. Further study will focus on TEAS activating the MC-nerve functional unit to regulate the pathophysiologic conditions.
Previous study identified that both CGRP and SP participated in MC-nerve associations 23 . In the present study, we found that TEAS activated the expression of both CGRP and SP in sensory nerves. The role of SP and its receptor on MCs were investigated in the local response to TEAS. Further study should be done to elucidate CGRP-MC crosstalk in local response of TEAS.
The procedure of MA therapy involves insertion and manipulation of the needles under skin to regulate Qi. The needles can enter either into muscles, near vascular walls or even into periostea to cause various feelings in patients 28 . Therefore, the local effects of acupuncture depend on the local tissues of acupoints as well as acupuncture manipulations, including twirling, lifting and thrusting needles. There are various approaches of acupuncture manipulations, and they are highly dependent on acupuncturist technique and subjective feelings of both doctors and patients. Generally, twisting at muscle-rich acupoints produces stronger distant effects than those produced   by cutaneous receptors 29 . Obviously, TEAS has limited clinical use. It is necessary to develop a new TEAS instrument that can replace acupuncture by mimicking manual manipulation in various tissues layers to achieve significant effects. Further studies on more reactions triggered by TEAS in various tissues are encouraged in the future, and more evidence is needed to prove whether EA or MA can be replaced by TEAS.

Conclusion
TEAS induced nerve fibres expressing neuropeptides CGRP and SP, SP activated the MC via binding to its NK-1R; activated MCs released 5-HT to convey acupuncture-like signals to certain pathways.

Data Availability
The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.