To establish a mouse model of chronic hyperalgesia, observe the analgesic effect of electroacupuncture, and explore the underlying mechanism of electroacupuncture analgesia from the perspective of inhibiting microglial activation in the spinal dorsal horn.

Eighty-three C57BL/6J mice were divided into a control group (n=26), a model group (n=27), and a electroacupuncture group (n=30) by complete randomization. Mice in the control group did not undergo any operation. Mice in the model and electroacupuncture groups received high-frequency, high-intensity electrical stimulation (frequency: 100 Hz, pulse width: 0.5 ms, intensity: 10 V) from the right sciatic nerve trunk to establish the chronic hyperalgesia model. The occurrence of pain in the behavior of mice was taken as the criterion for the successful establishment of the model. At Day 8 after modeling, electroacupuncture was performed on the right "Zusanli" (ST36) of the mice in the electroacupuncture group, once every other day for 32 consecutive days (20 min/session, 13 sessions in total). The myelin basic protein (MBP)in the sciatic nerve was assessed by immunofluorescence. The 50% paw withdrawal thresholds (50%PWT) and dynamic mechanical hypersensitivity (DMH) scores of the affected hind paw were measured with Von Frey filaments and a brush. Immunofluorescence was used to quantify ionized calcium-binding adaptor molecule 1 (Iba1) positive cells in the spinal dorsal horn. The expression of Iba1 protein in the dorsal horn was detected by western blotting. Inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-4, and IL-10 in the dorsal horn were quantified with fully automated nanoscale magnetic chemiluminescence.

Compared with the control group, no significant change in the MBP of the sciatic nerve in the model group mice was observed, whereas the 50%PWT dropped markedly and the DMH scores increased significantly (P < 0.01), indicating that a chronic hyperalgesia model with less peripheral nerve injury was successfully established. Compared with the model group, the 50%PWT of mice in the electroacupuncture group was higher and the DMH scores were lower (P < 0.05, P < 0.01), indicating that electroacupuncture can improve chronic hyperalgesia in mice. Compared with the model group, the mean fluorescence intensity of Iba1⁺ cells and the expression of Iba1 protein in the spinal dorsal horn of mice in the electroacupuncture group were decreased (P < 0.01), indicating that electroacupuncture inhibited the expression of microglia in the spinal dorsal horn of chronic hyperalgesia mice. Compared with the model group, the contents of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the spinal dorsal horn of mice in the electroacupuncture group decreased, and the content of the anti-inflammatory factor IL-4 was also decreased (P < 0.05, P < 0.01), but there was no significant change in IL-10. Taken together, these findings indicate that electroacupuncture can improve the inflammatory state of the dorsal horn of the spinal cord in mice.

High-frequency, high-intensity stimulation of the sciatic nerve can induce robust, long-lasting hyperalgesia without overt demyelination. Electroacupuncture at "Zusanli" (ST36) significantly alleviates this hyperalgesia, by suppressing spinal dorsal-horn microglial activation and modulating spinal cord dorsal-horn inflammatory status.

To observe the effect of electroacupuncture (EA) at “Zusanli” (ST36) on blood glucose in acute hyperglycemia (AH), low-risk prediabetes (LRP) and high-risk prediabetes (HRP) mice, and to explore its underlying mechanisms in regulating the function of pancreatic β-cells in LRP.

This study included 3 (AH, LRP and HRP) experiments. In each part of the experiments, C57 BL/6J mice were randomly divided into normal, model and EA groups, with 6 mice in each group in AH and HRP experiments, and 18 mice in LRP experiment. The AH model was established by intraperitoneal injection of 2 g/kg glucose solution. After injection, EA (1 mA, 10 Hz) was applied to bilateral “Zusanli” (ST36) for 20 min. The LRP model was established by feeding the mice with high-fat diet for 1 week. From the 4^th day when the impaired glucose tolerance criteria of prediabetes were reached, EA was applied to bilateral ST36, with the parameters being the same as above, once a day for a total of 4 times. In the HRP experiment, the animal model was established by feeding the mice with a high-fat diet for 8 weeks. Four weeks after beginning of the modeling, EA intervention (1 mA, 10 Hz) was performed at ST36 for 20 min, 3 times a week for a total of 12 times. Blood glucose and glucose tolerance were measured using a glucose meter, and serum glycated hemoglobin A1c (HbA1c), insulin, and glucagon levels were detected by enzyme-linked immunosorbent assay (ELISA). Changes of the pancreatic morphology were examined by HE staining. The expression levels of activating transcription factor 6 (ATF6), endoplasmic reticulum to nucleus signaling 1 (ERN1), eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2 AK3), p21 (a cell cycle factor in cellular senescence), muscarinic acetylcholine receptor M2 (M2-AchR) and M3-AchR in the pancreatic β-cells were detected using Western blot, and the expression levels of pancreatic and duodenal homeobox factor-1 (PDX-1), beta-cell-specific transcription factor (MAFA) and forkhead box O1 (FOXO1) mRNAs in the pancreatic β-cells detected using quantitative real-time PCR. The insulin⁺/P21⁺ co-positive cells, and the number of ChAT⁺/c-fos⁺ co-labeled neurons in the pancreatic ganglia and CTB647/ChAT⁺/c-fos⁺ co-labeled neurons in the dorsal motor nucleus of the vagus (DMV) innervating the pancreas were determined using fluorescent immunohistochemistry.

1) In the AH experiment, compared to the normal group, the blood glucose level in the model group was significantly increased 15 min after modeling (P<0.001), without pathological damage in the pancreas, and the serum insulin level was significantly increased after modeling (P<0.05). In comparison with the model group, the blood glucose levels at 30 and 60 min were significantly reduced (all P<0.01), while the serum insulin content was considerably increased (P<0.05) in the EA group, suggesting that EA lowered blood glucose by increasing stress-induced insulin level. 2) In the LRP experiment, compared to the normal group, the blood glucose levels from the 3^rd day to the 7^th day, and the glucose tolerance from 15 to 120 min were significantly elevated in the model group (P<0.001, P<0.05, P<0.01). No significant changes were found in the serum HbA1c, serum insulin and glucagon levels and in the morphological result of the pancreas tissue in the model group. In comparison with the model group, the blood glucose levels on day 4, 6 and 7, the glucose tolerance at 15, 30, 60 and 120 min were strikingly decreased (P<0.001, P<0.01, P<0.05), while the serum insulin content was notably increased (P<0.05) in the EA group. No significant changes were found in the serum HbA1c and glucagon levels after EA intervention. 3) In the HRP experiment, compared to the normal group, the blood glucose levels from 1 to 8 weeks, the glucose tolerance levels at 15, 30, 60 and 120 min, HbA1c and insulin levels were significantly increased (P<0.001, P<0.05, P<0.01), whereas the serum glucagon level was notably decreased (P<0.05) in the model group, accompanied with an increase of the pancreatic islet volume. Following EA intervention, the blood glucose at the 5^th and 6^th week, insulin and glucagon levels were considerably down-regulated in the EA group (P<0.01, P<0.05), accompanied with a slight decrease of the pancreatic islet volume. 4) In LRP mice, in comparison with the normal group, the expression levels of M2-AchR and M3-AchR protein were significantly decreased (P<0.05), while the expression levels of ATF6, ERN1, EIF2 AK3, P21 and the number of insulin⁺/P21⁺ positive cells in the pancreas tissue were significantly increased (P<0.01, P<0.05) in the model group. In contrast to the model group, the EA group had an obvious increase in the number of CTB647/ChAT⁺/c-fos⁺ co-labeled neurons in the DMV and ChAT⁺/c-fos⁺ co-labeled neurons in the pancreatic ganglia, and the expression of M2-AchR and M3-AchR protein (P<0.05, P<0.01), and a notable down-regulation in the expression levels of ATF6, ERN1, EIF2 AK3 and P21 proteins and in the number of pancreatic insulin⁺/P21⁺ positive cells (P<0.01, P<0.05). No significant changes were found in the number of CTB647/ChAT⁺/c-fos⁺ co-labeled neurons in the DMV and ChAT⁺/c-fos⁺ co-labeled neurons in the pancreatic ganglia in the model group, and in the mRNA expression levels of pancreatic PDX1, MAFA and FOXO1 in both model and EA groups.

EA at ST36 can promote the restoration of blood glucose balance in the AH, LRP and HRP stages in mice, with the most effective phase being the LRP. The underlying mechanism may be related to its functions in activating vagus nerve to inhibit the endoplasmic reticulum stress in pancreatic β-cells.

To observe the differential analgesic effects of electroacupuncture (EA) at different intensities on calcium activity in dorsal root ganglia (DRG) neurons induced by mechanical, thermal, and cold stimuli in a state of muscular inflammatory pain, so as to preliminarily reveal the peripheral integration characteristics of acupuncture analgesia mechanisms.

The inflammatory muscle pain model was established by injection of complete Freund’s adjuvant (CFA) into the right tibialis anterior muscle. EA (0.5 mA or 1 mA, 2 Hz) was applied to “Zusanli” (ST36) on the right side for 10 min. Before and after the EA, mice were given body surface stimulation of brush, clamp and different temperature, while simultaneously recording DRG neuron calcium activity during stimulation. The effects of different stimulation methods before and after EA on the change in fluorescence intensity (ΔF/F0) and the percentage of activated DRG neurons were compared.

In the clamp hind paw stimulation, both 0.5 mA and 1 mA EA could reduce the fluorescence intensity of neurons, and the effect of 1 mA was better than that of 0.5 mA (P<0.001). Specifically, 0.5 mA EA decreased the percentage of medium and large neurons (P<0.05), while 1 mA EA decreased the percentage of small, medium, and large neurons (P<0.05, P<0.01), and the effect of 1 mA was better than that of 0.5 mA in reducing the percentage of small and medium neurons (P<0.01). In the stimulation of clamping tibialis anterior muscle, both EA intensities could reduce the fluorescence intensity of neurons (P<0.05, P<0.0001), and the effect of 1 mA was better than that of 0.5 mA (P<0.0001), and 1 mA EA could reduce the percentage of small and medium neurons (P<0.05). In the brush stimulation, both EA intensities could reduce the fluorescence intensity of neurons (P<0.0001), and the effect of 1 mA was better than that of 0.5 mA (P<0.0001), and 0.5 mA EA decreased the percentage of medium and large neurons (P<0.05), while 1 mA EA decreased the percentage of small, medium and large neurons (P<0.05, P<0.01). However, in the temperature stimulation (43, 51, 4 ℃), there were no obvious changes in the fluorescence intensity and the percentage of activated neurons in different intensities of EA.

Both 0.5 mA and 1 mA EA intervention at ST36 can inhibit the reactivity of DRG neurons to mechanical stimulation, with higher intensity EA showing more significant effects.

To observe the effect of electroacupuncture (EA) at “Zusanli” (ST36) on improving cardiac function in mice with chronic heart failure (CHF), so as to explore the mechanism of its regulation on the autonomic nervous system.

Eighteen C57BL/6J mice were randomly divided into sham-surgery, model, and EA groups, with 6 mice in each group. The model of myocardial ischemia followed by CHF was induced by ligation of the anterior descending branch of the left coronary artery. Two weeks after modeling surgery, EA intervention (0.5 mA, 2 Hz/15 Hz) was performed bilaterally at ST36 in the EA group, 20 min each time, once every other day for 4 weeks. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVESD), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-diastolic volume (LVEDV) were evaluated using echocardiographic imaging. The exercise tolerance was observed in an exhaustive test. Electrocardiograms were recorded using an electrophysiological recording system to analyze heart rate (HR) changes before and after EA, and frequency domain analysis was used to analyze the heart rate variability (HRV), including low-frequency (LF) component, high-frequency (HF) component, and their ratio (LF/HF). Immunofluorescence was used to observe the number of choline acetyltransferase (ChAT)⁺/c-fos⁺ co-labeled neurons in the dorsal motor nucleus of the vagus (DMV).

In terms of cardiac function, compared with the sham-surgery group, the LVEF and LVFS were decreased (P＜0.001) and the LVESD, LVEDD, LVESV, and LVEDV were increased (P＜0.001, P＜0.01) two weeks after modeling of mice in the model group. After 2 weeks of EA intervention, the LVESD and LVESV in the EA group were significantly smaller than those in the model group (P＜0.05). After 4 weeks of intervention, the LVEF and LVFS in the EA group were significantly higher than those in the model group (P＜0.05), and the LVESD and LVESV were significantly reduced (P＜0.05, P＜0.01). Compared with the model group, EA significantly improved the exercise tolerance of CHF mice (P＜0.05). Compared with the sham-surgery group, the LF/HF in the model group and EA group were significantly increased (P＜0.05) 2 weeks after modeling, indicating autonomic imbalance. After 4 weeks of intervention, the LF/HF in the EA group showed lower trend than that in the model group but the difference was not statistically significant. Additionally, after immediate EA, HR decreased (P＜0.05, P＜0.01), HF increased (P＜0.01), and both LF and LF/HF decreased significantly (P＜0.05) of mice in the EA group, indicating that EA corrected the autonomic imbalance in CHF mice. The number of ChAT⁺/c-fos⁺ co-labeled neurons in the DMV of the EA group was significantly higher (P＜0.001) than that in the model group.

EA at ST36 significantly improved cardiac function and exercise tolerance in CHF mice. The underlying mechanism may be related to the activation of vagus efferent nerve and the regulation of autonomic nerve balance.

To observe the effect of electroacupuncture (EA) on the neuronal activity of primary afferent dorsal root ganglion (DRG) caused by different mechanical stimuli such as brush, clamp [clamping hind paw or "Zusanli" (ST36) acupoint] or stimulation at different temperatures (43 ℃, 51 ℃, 4 ℃), so as to explore the interaction between EA signals and different primary afferent signals at the DRG level.

The strength threshold of activated A fiber was determined by electrophysiological technique. DRG neurons were labeled by intrathecal injection of AAV9 virus, and the response of DRG neurons to different primary afferent stimuli was observed and recorded by calcium imaging. EA (0.5 mA, 0.2 ms, 2 Hz) was applied to "Zusanli" (ST36) on the right side for 10 min. The changes of the number, percentage of neurons of different sizes and fluorescence intensity of DRG neurons activated under different stimulation before and after EA were compared.

Clamping stimulation, brush stimulation, or different temperature stimulation could all activate DRG neurons of large, medium, and small classes. EA at ST36 could significantly decrease the percentage of activated small neurons and large neurons in DRG induced by clamping the hind paw (P<0.05), and reduce the fluorescence intensity of neurons with different diameters (P<0.0001), suggesting that EA could suppress the neuronal activity induced by noxious mechanical stimulation of the hind paw. However, EA at ST36 had no significant effect on the percentage of activated DRG neurons and fluorescence intensity caused by clamping the anterior tibial muscle. Meanwhile, EA at ST36 could significantly reduce the percentage of activated neuron in DRG induced by brush stimulation (P<0.05), and reduce the fluorescence intensity of neurons with different diameters (P<0.0001). In temperature stimulation, EA could significantly inhibit the fluorescence intensity of DRG neurons under 4 ℃ cold stimulation (P<0.01), but had no significant effect on the percentage and fluorescence intensity of DRG neurons activated by 43 ℃ and 51 ℃.

EA at ST36 can inhibit the nociceptive mechanical input of the hind paw and the input of 4 ℃ cold stimulation.