Effect of Electroacupuncture on 99mTc-Sodium Pertechnetate Uptake and Extracellular Fluid Free Molecules in the Stomach in Acupoint ST36 and ST39

Electroacupuncture (EA) is a therapeutic modality in which the electrical stimulation is integrated with concepts of acupuncture to treat diseases. This study was designed to evaluate the connection between the electro-acupuncture induced increase in Na99mTcO4 uptake in the stomach wall, and the ionic molecule levels in the extracellular fluid in the acupoints. Wistar rats were treated by 2 or 100 Hz EA at Zusanli (ST 36) and Xiajuxu (ST 39) bilaterally for 60 minutes. The accumulation of Na99mTcO4 in the gastric wall and the free ions, including Ca2+, K+, Na+, and Cl−, in the acupoints were measured every 60 minutes. The radioactivity uptake in the stomach was significantly increased during EA, reaching peak at 180 minutes after the EA. The concentration of extracellular ions was also significantly increased during EA. The Ca2+ level continued to rise until 60 minutes after EA, then started to decrease at 120 minutes post-EA. The results suggest this up-regulatory effect of EA on gastric activity might be triggered by the increase of the extracellular ion levels, this effect lasts longer than stimulating the release of transmembrane Ca2+ flow alone. This might aid in providing a better understanding of the long-lasting effect claimed in acupuncture treatment.


Electroacupuncture treatment. Rats were anaesthetized with an intraperitoneal injection of 20%
Urethane at a dose of 5 mg/kg. Rats were placed in a ventral decubitus position. EA was applied at Zusanli (ST 36) and Xiajuxu (ST 39) bilaterally as previously described 12 . EA was given for a total of 60 minutes (1 mA stepwise to 3 mA, 0.2 ms duration, 2 or 100 Hz). Rats in the non-EA group were also anaesthetised in a similar fashion for 60 minutes as described 12 . Microdialysis system components, target molecule detection, and calculation. The CMA 30 Linear Microdialysis Probe (CMA Microdialysis, Stockholm, Sweden) to study the peripheral tissues (diameter 0.24 mm, length 21 cm, membrane length 10 mm) was used. The probe consists of inlet tubing, outlet tubing, and a middle part with a window where the membrane is located. The inlet of the probe was connected to a peristaltic pump (CMA Microdialysis, Stockholm, Sweden), which would push the tube with normal saline (NS) at a rate of 2.0 µL/min. Before sample collection, the membrane part of the probe was immersed in distilled water for 30 minutes. Then, the probe was immersed in the tissue around ST 36 (depth of about 3 mm), and the dialysate was collected in the outlet part every 60 minutes for the following 4 hours (i.e., before EA, during EA, 60 minutes after EA, and 120 min after EA). The dialysate was stored at −20 °C for further analysis.
We tested the target molecules, detecting efficiency of the probe with standard ion solutions. The MD probe was put into NS for 20 minutes for stabilisation and then was switched to standard ion solutions. The standard ion sampling was collected at 60-minute intervals. All diluted dialysates samples were analysed for Ca 2+ , Na + , K + , and Cl − concentrations by LABOSPECT008 (Hitachi Japan). Dialysate ions were calculated as 13 : Na 99m TcO4 imaging of the stomach wall. Na 99m TcO4 SPECT/CT was performed 5 minutes after the injection of 7.4 MBq of 99m TcO4 − via the caudal vein. The scanners were dual-head gamma cameras, using low-energy high-resolution collimators and a 20% energy window centred on 140 keV. The DICOM image files of each rat were saved on optic discs and transferred to a Symbia Evo Excel workstation (Siemens Healthineers) for centralized reconstruction, reading, and analysis. Na 99m TcO4 SPECT/CT images were read and interpreted by the consensus of three experienced nuclear medicine physicians with reference to SPECT/CT fusion images. For semiquantitative analysis, gastric-to-background ratios of SPECT images were measured and calculated by the same person using a consistent standard 14 . The data are collected and compared with before EA, during EA, and after EA, to observe the adjusting effect of EA on gastric function. Statistical analysis. The data were analysed using SPSS 10.0 software (SPSS Inc. Chicago, Ill., USA). All results are expressed as the mean ± standard error (SEM). Two-way ANOVA and Duncan post hoc tests were performed to investigate the effects of electroacupuncture on the extracellular ions concentrations and on the gastric functions during each session, and a p-value < 0.05 was considered statistically significant.
Availability of data and materials. The data sets used and/or analyzed during this study are available from thecorresponding author upon reasonable request and have been approved by the institutional committee on scientific research. Ethics approval. This study is approved by the ethics committee on biomedical study of the Xi'an Jiaotong University Health Science Center.

Results
The Ions levels at ST 36 increased significantly during EA. As reported previously, the target molecules detecting efficiency of the probe was evaluated with the standard solutions of Ca 2+ , K + , Na + , and Cl − (Table 1).
Stimulating ST 36 with 2Hz-EA increased the concentration of extracellular Ca 2+ significantly during EA (p = 0.003 vs pre-EA). The Ca 2+ level increased gradually and reached the peak plateau at 60 minutes after 2Hz-EA (p = 0.75 vs EA), then started to decrease at 120 minutes post-EA (p = 0.04 vs EA). The concentrations of extracellular Na + and Cl − were also significantly increased during EA as compared with pre-EA (p < 0.001 and p = 0.007, respectively). After EA, they started to decrease. Their levels decreased to 71.81 ± 15.09 mmol/L and 57.42 ± 14.30 mmol/L 60 minutes after 2Hz-EA (p = 0.09 and p = 0.07 vs. EA). The level of extracellular K + showed similar changes, although the comparisons showed no significance (Table 2).  Compared with the 2 Hz group, EA at 100 Hz also significantly enhanced the increase of the extracellular Ca 2+ , Na + , and K + concentrations during EA, and the effect did not disappear 60 minutes after EA. However, no significant difference was found between the two groups ( Table 2, Fig. 2).

Concentrations detected
The radioactivity uptake in the stomach was significantly upregulated during EA. Compared with before EA, the radioactivity uptake in the stomach was significantly increased during 2 Hz-EA (T/B ratio, 10.98 ± 0.75 mmol/L vs 8.96 ± 0.99 mmol/L). Interestingly, the up-regulation increased gradually (T/B ratio, 14.74 ± 0.46 mmol/L and 18.72 ± 0.84 mmol/L at 60 min 120 min after EA, respectively) and did not reach the peak until 180 minutes after the EA (T/B ratio 22.80 ± 1.40 mmol/L at 180 min after EA). Then the stomach uptake started to slowly decrease 3 hours after 2 Hz EA at ST 36 (Fig. 3).
100 Hz EA appeared to induce more radioactivity uptake than did 2 Hz EA in the stomach; however, no significance was reached when comparing the two groups (Fig. 4). The Ca 2+ level continued to increase till 60 minutes after EA, and then started to decrease in post-EA at 120 minutes (p = 0.04 vs. EA). Similar to the situation under 2 Hz, the extracellular Ca 2+ concentrations also increased significantly under high frequency (100 Hz) EA, and the increase started to decrease 120 minutes after EA. The difference between low and high frequencies (2 vs 100 Hz) did not reach significance. Compared with extracellular Ca 2+ levels during EA, *p < 0.05, **p < 0.01, Two-way ANOVA followed by Duncan post hoc tests. The unbound form of pertechnetate anion (Na 99m TcO4) is distributed throughout the vasculature and interstitial fluids by a slow diffusion mechanism, and the concentration varies between organs 15 . In the gastrointestinal tract, Na 99m TcO4 is actively concentrated mainly in the gastric mucosa 16 . The bioavailability of Na 99m TcO4 in the gastric wall can be changed in many circumstances, including the patient's health status, disease states, invasive medical procedures, and complementary therapies such as acupuncture 17 . Despite that the specificity of acupuncture is still questioned, the actions of acupuncture at ST 36 have been shown to significantly increase the radioactivity uptake of Na 99m TcO4 in the stomach 9 . In the current study, we utilized the Single positron emission tomography (SPECT/CT) as main testing modality to observe the gastric function under electroacupuncture (EA) in vivo. (SPECT/CT) could reproduce anatomical and physiological imaging by mapping GIT changes in vivo in response to acupuncture stimuli 18 . Our results suggest the up-regulation effect of EA on gastric wall Na 99m TcO4 uptake. Previous studies have suggested that EA could produce an excitatory effect on the gastrointestinal motility of the rat 9 . These ST 36 acupuncture effects may be correlated with our data, which suggested that 2 Hz-EA could obviously increase the uptake of Na 99m TcO4 in the stomach, whereas no significant differences were found between the 100Hz-EA group and the control group.
Compared with simple acupuncture, EA is a therapy in which the principles of electrical stimulation are integrated with traditional acupuncture 6 . The two electrical frequencies 100 Hz (high frequency) and 2 Hz (low frequency) are commonly used for EA research 19 . Our results showed that EA at both frequencies could induce increases in gastric function, similar to previous studies. Tian et al. suggested that EA at both low and high frequencies could induce a therapeutic effect on obesity 20 . Tseng et al. showed that EA in either frequency could induce a decrease in glucose, an increase in lactate metabolites, and a reduction in lactate/glucose ratios 21 . Studies of EA in which ST 36 has undergone electrostimulation at 2 or 100 Hz have suggested that the mechanism underlying the effect might be that EA could lead to the regulation of internal organ systems as a network 22 . EA stimulation could lead to effects on gastric myoelectrical activity, brain-gut peptides responses in the gastric mucosa, and the vagal and splanchnic nerve responses on the activity of gastric motility and emptying 23 .
In recent years, more and more laboratory proof has accumulated that acupuncture can change the charge and potential of neurons, the concentrations of K + , Na + , Ca 2+ , and the content of neurotransmitters [3][4][5] . Among them, Ca 2+ was supposed to play a key role 5 . As an important intracellular second messenger, Ca 2+ participates in various physiological and biochemical processes of cells 24 . The proposed acoustic signaling paths, jing-luo, are distinctly and physically separated from the nervous system. This appears to be the missing "calcium" communication link proposed but not found in the frequency-encoded second messenger 25 . It has been suggested that calcium may be taken as the carrier of the biological modulation system, of which acupuncture has been implemented as a clinical application 26 . Moreover, because the intracellular Ca 2+ concentration is controlled by the opening and closing of both voltage-dependent and receptor-dependent Ca 2+ pathways, as well as by the Na + /Ca 2+ exchanger and the activity of the Ca 2+ pump, the concentrations of K + and Na + were reported to change with Ca 2+ levels 27 .
The effects of acupuncture at the points of the Foot-Yangming meridian on gastric movement are related to the release of intracellular Ca 2+ in the gastric smooth muscles 1 . The AM and FM schemes of calcium signaling in the acupuncture was proposed to cause cellular Ca 2+ flux. The signal is broadcasted with a chevron-like pattern radiating from the needle like a radio antenna 28 . Our study showed that acupuncture at ST 36 and ST 39 could markedly enhance release of intracellular Ca 2+ and, thus, significantly increased the extracellular Ca 2+ levels. The extracellular Figure 4. Compared with pre-EA, the radioactivity uptake in the stomach was significantly increased during 2 Hz-EA. The up-regulation escalated gradually and did not reach the peak until 180 minutes after the EA. The stomach uptake started to decrease 3 hours after 2 Hz EA at ST 36. 100 Hz-EA appeared to induce more radioactivity uptake than did 2 Hz EA in the stomach; however, no significance was reached in comparison of the two groups. Compared with radioactivity uptake in the stomach during EA, *p < 0.05, **p < 0.01, Two-way ANOVA followed by Duncan post hoc tests. Ca 2+ level increase was correlated with gastric function upregulation. One potential testable hypothesis could be that the AM and FM schemes of calcium signaling were proposed based on the amplitude modulation of extracellular Ca 2+ levels, and separated from the central nervous system, there might be a channel of cellular communications with calcium waves playing the role 3 . This proposed mechanism might explain our surprising finding that the increase of the gastric wall Na 99m TcO4 uptake lasted longer than the enhancing of the release of intracellular Ca 2+ to extracellular space observed in our study. Similar observations have been reported that Ca 2+ oscillation remained at 30 minutes or even 1.5 hours after the needle stimulation was turned off 5 . The latency and memory effects appeared to agree with the long-lasting healing claimed in traditional acupuncture treatment 29 .
In this experimental model, the authors did not include the "sham acupuncture" group as an "inactive" control, since needling non-acupoints (any cutaneous location of the body surface) could produce unpredictable somatovisceral reflex responses, and therefore could complicate the interpretation of the results 30 . Our experimental finding is that the when EA was applied at non-acupoints, the coupling of the increase of Ca 2+ levels and gastric Na 99m TcO4 uptake disappeared. There is a slight increase in the Ca 2+ level, whereas no upregulation of the gastric function was observed (data not shown).
In the current study, EA at acupoints of the stomach meridian in rats showed that EA could upregulate gastric function, which is thought to be correlated with extracellular ions concentrations changes at the acupoints. Further rigorous experimental studies to examine their correlations need to be performed.