All experimental procedures were conducted following the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (protocol code, DC1237, 01 January 2019). The experiments were performed on male Sprague–Dawley (SD) rats (weighing 220–250 g) aged 6–8 weeks that were purchased from Chengdu Dossy Experimental Animals Co., Ltd. Animals were housed at standard laboratory conditions (24 ± 2°C room temperature and 65 ± 5% relative humidity on 12/12 h conventional light–dark cycles) and fed with standard laboratory chow and tap water ad libitum. After adaptive domestication for 1 week, mice were divided into different groups based on random numbers generated by the IBM SPSS Statistic 25 software, and assigned to individual mice.

EA stimulation was administered by using an electroacupuncture apparatus (HANS-200A Acupoint Nerve Stimulator, Nanjing Jisheng Medical Technology Co., Ltd., Jiangsu, China) at roughly the same time of the day (10:00 a.m. to 12:00 p.m.). One stainless-steel acupuncture needle (0.25 × 25 mm, Hwato-Med. Co., Jiangsu, China) was inserted into the left “Zusanli” acupoint (ST36), located about 6 mm down from the left fibular head, with a depth of 5–8 mm (Figure 1A). Another needle was applied to the region without acupoints, namely the stump of the tail. The position of ST36 in rats corresponds anatomically to its location in humans. For sham EA at ST36, the needle was inserted 2–3 mm deep into the skin dermal tissue and left there for 30 min, without any electrical stimulation. In the case of EA, the negative output of the stimulator was connected to the needle at ST36, and the auxiliary needle was connected to the positive output of the stimulator. The electrical current range was set at 1 mA, with a frequency of 15 Hz for 30 min (Figure 1A). The rats were immobilized by a self-made device during treatment (28).

The pH of PBS (Sigma-Aldrich, Shanghai, China) was adjusted to 6.0 using acetic acid (Sigma-Aldrich) and sodium hydroxide (Sigma-Aldrich). 100 μL of PBS (pH 6.0) was injected into the left hind paw of SD rats to induce plantar hyperalgesia. Plantar pain threshold was determined as thermal withdrawal latency (TWL) by using a Thermal Stimuli Instrument (PL-200, Techman Software Co., Chengdu, China). The plantar surface of the left hind paw responds to thermal laser stimulation so that withdrawal, shaking, or licking of the left hind foot becomes apparent. Each rat was tested six times, with intervals of 5 min, every 30 min, including the following six points-in-time: −30 (baseline), as well as 0, 30, 60, 90, and 120 min. pH 6.0 PBS was injected at the point of 0 min (Figure 1A). All rats were placed separately into a transparent plastic enclosure (210 mm × 210 mm × 160 mm) on the surface of a vitreous platform (800 mm × 400 mm × 165 mm) for 30 min every day to get accustomed to the experimental conditions, 3 days before behavioral testing. All behavioral data were recorded by the same investigator who was blind to the experimental grouping. Rats with less than a 30% decrease in pain threshold at time-point 0-min were removed.

The SD rats were anesthetized with isoflurane (5% for induction; 2% for maintenance; RWD Life Science, San Diego, CA, USA) and their head was fixed on a stereotaxic platform (RWD Life Science). 0.5 μL of adeno-associated virus (AAV) in a glass syringe was injected bilaterally into the S1HL (stereotaxic coordinates: AP -1.2 mm, ML ± 3.0 mm, DV -2.0 mm; see Figure 2B) at a rate of 0.05 μL/min with a microsyringe pump (RWD Life Science). An additional 10 min were allowed for diffusion and prevention of backflow. At the end of the surgery, 5 mg/kg enrofloxacin (RWD Life Science) was administered subcutaneously to the animals to prevent postoperative infection, and all animals were placed on heating pads (37°C) during surgery to keep their body temperature stable.

For chemogenetic manipulation, the following viruses were used in this study: rAAV-GfaABC1D-hM4D(Gi)-EGFP (titer: 2.02 × 10¹² VG/mL, AAV2/5), rAAV-GfaABC1D-hM3D(Gq)-EGFP (titer: 2.10 × 10¹² VG/mL, AAV2/5), rAAV-GfaABC1D-EGFP (titer: 2.10 × 10¹² VG/mL, AAV2/5, all from Brain Case, China). The specific application mode of the virus is documented in Supplementary Table 1. The expression of the virus was checked by immunofluorescence staining after all tests were finished.

At least 3 weeks were allowed to pass for the complete expression of the virus. The activity of astrocytes was modulated by the chemogenetic receptors hM3Dq (Gq) and hM4Di (Gi) (see Supplementary Table 1 for specific manipulations). Next, rats were intraperitoneally injected with 1 mg/kg of clozapine N-oxide (CNO; Sigma-Aldrich, Saint Louis, MO, USA) to activate Gq and Gi receptors and the baseline of TWL was measured 30 min later. The pH 6.0 PBS was injected into the left paw of the hindlimb after obtaining the TWL (baseline), which was determined subsequently every 30 min until the time point of 120-min (Figure 2A).

SD rats were anesthetized with 2% pentobarbital sodium (40 mg/kg; Sigma-Aldrich) and transcardially perfused with 200 mL of 0.9% NaCl followed by 4% paraformaldehyde (PFA). Their brains were prepared and fixed in 4% PFA for 24 h, then they were stored at −80°C after gradient dehydration. The collected tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek, Umkirch, Germany) and cut into 15-μm-thick sections with a freezing microtome (CM1806, Leica, Zurich, Switzerland); then they were incubated with the following antibodies: Mouse anti-GFAP antibody (1: 200, Proteintech Group, Chicago, USA), Goat Anti-Mouse IgG H&L (1: 400, Bioss, Beijing, China), Goat Anti-Mouse IgG H&L/Cy3 (1: 400, Bioss). Images were acquired using a confocal laser scanning microscope (Zeiss LSM700, Oberkochen, Germany) and quantified using Image J software. The quantity of GFAP immunoreactivity was expressed as GFAP-positive area in percentage of the total area in which GFAP-labelled immunoreactivity was determined.

All values were expressed as Mean ± S.E.M. (standard error of means). The data were analyzed and plotted using IBM SPSS Statistic 25 and GraphPad Prism 9. The normality test was conducted in IBM SPSS Statistic 25; all data were normally distributed. Multiple comparisons of data were performed with GraphPad Prism 9. Multiple groups were compared by one-way ANOVA followed by the Bonferroni post-hoc test. The statistical significance was defined as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; *p ≤ 0.05 was considered statistically significant.

To assess the analgesic effects of EA, we used the plantar injection of pH 6.0 PBS in the left hindlimb of SD rats to establish an acid-induced pain model and used the TWL test for 120 min to measure the change of pain threshold during this time (Figure 1A). We found that every group (normal PBS, pH 6.0 PBS, PBS + EA, pH 6.0 + EA) showed a similar baseline of the TWL at the −30 min time-point (Figures 1B,E). The acidic PBS caused a pronounced fall in the TWL value, 30 min after injection to the left paw that remained stable for 120 min, although the low pH PBS was expected to become diluted constantly in the tissue; by contrast, the injection of PBS at a normal pH of 7.4 had no impact on the TWL (Figures 1B,C). The results suggested that plantar injection of pH 6.0 PBS induced acute thermal hyperalgesia, and the acid-induced pain model was successfully established.

EA treatment was applied to the ipsilateral acupoint ST36 for 30 min after injecting normal or acidic PBS (Figures 1A–D). EA abolished the effect of pH 6.0 PBS at 0 min and this antagonism continuously vanished throughout the following 120-min, when it finally was no longer apparent (Figure 1B). By contrast, EA had no effect on the TWL measured after the injection of normal PBS. Figure 1C shows that the application of pH 6.0 PBS had the same effect at 0 and 120 min when expressed as the ratio of TWL and its baseline value at these two time points. In contrast, the TWL ratio measured at 0-min did not change when EA was applied in combination with acidic PBS. Nonetheless, this effect of EA was only temporary, and completely disappeared at the 120 min time point (Figure 1D). We also investigated the effect of sham EA, and found that this treatment failed to alter the effect of the acidic PBS on the TWL (Figures 1E,F).

GFAP is the most widely used biomarker of reactive astrocytes (29). To investigate the influence of EA treatment on astrocytic activation, we conducted immunofluorescence staining and quantified the expression of GFAP in the contralateral (right) S1HL region after injection of normal and acidic PBS, applied in combination of the latter with EA or sham EA (Figure 3A). The injection of pH 6.0 PBS into the left hind paw did not change the amount of GFAP (percentage of GFAP area; see Methods), when compared with that measured after the injection of normal pH PBS (Figures 3A,B). In contrast, the application of EA after low pH stimulation, markedly increased the amount of GFAP staining (Figure 3B), probably indicating the activation of contralateral astrocytes and the consequent development of astrogliosis. The combination of pH 6.0 PBS with sham EA had no comparable effect, although the tendency of the amount of GFAP staining to increase might be due to the mechanical stimulation of subcutaneous tissue in the acupoint by the needle, without delivering an accompanying electrical current.

We next examined whether astrocytes regulated the pH 6.0-induced pain. Immunmohistochemistry showed that the chemogenetic receptors were in fact expressed in GFAP-positive astrocytes in the S1HL region within the different experimental groups (Figure 2B).

The graphs illustrate that the TWL baselines were not modified when either Gq or Gi receptors were expressed in rat brains and were afterwards activated by intraperitoneal injection of CNO (1 mg/kg) (Figures 2C,E). The stimulation of S1HL astrocytes via Gq activation failed to restore the normal TWL but slightly prolonged the time until the onset of the pH 6.0-induced paw withdrawal from thermal stimulation (Figure 2C). In partial disagreement with these findings, abolishing the influence of S1HL astrocytes by Gi-mediated inhibition did not produce any change of TWL in rats caused by acidic PBS (Figure 2E). The respective control measurement in rats whose S1HL region was infected with the EGFP-carrying rAAV, which however lacked the Gq or Gi components, did not interfere with the pain-inducing effect of normal or acidic PBS (Figures 2C,E). This becomes still better visible, when the percentage TWL/baseline ratios are being considered, as documented in the Figures 2D,F.

We have proven that the pH 6.0-induced hyperalgesia could be strongly suppressed by EA stimulation and rather moderately by astrocytic activation via Gq receptors alone. Considering the positive link between EA and astrocytes that was found in previous work (26, 27) we directed our attention to the relationship between EA-induced analgesia and astrocytic functions. To illustrate the involvement of the S1HL astrocytes in the pain control of EA stimulation, EA was applied for 30 min after activating the chemogenetic receptors Gq or Gi by CNO in the pH 6.0-induced pain model of rats.

The respective TWLs show that EA applied to rats with expressed Gq receptors in cortical astrocytes and added CNO, strongly potentiated the effect of EA (Figure 4A). This was evident also when the TWL/baseline ratios were taken into consideration (Figure 4B). To further elucidate the role of S1HL astrocytes, we repeated our experiments after inhibiting these astrocytes through the stimulation of their expressed Gi receptors by CNO. We found that inhibition of the activity of S1HL astrocytes conspicuously counteracted the analgesic effect of EA, whereas CNO administration to rats without available chemogenetic receptors (only EGFP present) was not able to remove this effect of EA (Figure 4C). As already pointed out, it is quite clear that the analgesic effect of EA was maximal at the time point of 0-min, and then gradually diminished until it disappeared at the time point of 120-min. Correspondingly, the antagonistic effect of astrocytic inhibition via Gi on EA effects was the largest at 0-min and totally vanished at 120-min (Figure 4D). This observation emphasized the involvement of astrocytes in EA-induced analgesia of acidic thermal hypersensitivity. Thus, the Gi-mediated inhibition of S1HL astrocytes suppressed, whereas the Gq- mediated activation potentiated the analgesic effect of EA against acid-induced pain.