Electro-acupuncture at “Zusanli” (ST36) alleviates anxiety and pain in mice with chronic inflammatory pain-anxiety comorbidity

To observe the effect of electro-acupuncture (EA) at different acupoints on chronic inflammatory pain-anxiety comorbidity in mice, and to investigate the effect of naloxone on EA induced activation of neurons of the anterior cingulate cortex (ACC), medial prefrontal cortex (mPFC), paraventricular nucleus of the thalamus (PVT), and mediodorsal thalamus nucleus (MD), so as to explore whether the anti-anxiety effect of EA depends on its analgesic mechanism.

In the first part of the experiment, the efficacy of EA at different acupoints was compared. Sixty male C57BL/6 mice were randomly divided into control, model, EA Zusanli (ST36), EA Baihui (GV20), and EA Shengmen (HT7) groups (12 mice/group). In the second part, the impact of naloxone on the analgesic and anxiety effects of EA at ST36 was investigated. Forty male C57 BL/6 mice were randomly divided into control, model, EA ST36, and EA ST36 + naloxone groups (10 mice/group). Except for those in the control group, all the mice were subjected to a chronic inflammatory pain-anxiety comorbidity by subcutaneous injection of 20 μL emulsified complete Freund’s adjuvant (CFA) into the hind paw. Mice in the control group received an equivalent volume of PBS. EA (2 Hz/100 Hz, 0.2—0.4 mA) intervention was initiated on the 12^th day after modeling, and applied to “Zusanli” (ST36, bilateral) or “Baihui” (GV20), or “Shengmen” (HT7, bilateral). The EA intervention was conducted for 30 min, once daily for 6 d. Naloxone was intraperitoneally injected 30 min before EA intervention. Paw withdrawal thresholds (PWTs) were measured using von Frey filaments. The anxiety-like behaviors were assessed using the open field (OF) test and elevated plus maze (EPM) test, separately. The expression of c-Fos in the ACC, mPFC, PVT, and MD was detected by immunofluorescence staining.

Compared with the control group, the model group exhibited a significant decrease in the PWTs, number of entries into the open arms and time spent in the open arms in the EPM test, and in the number of entries into the central area, time spent in the central area, and distance traveled in the central area in the OF test (P<0.01), and a significant increase in the number of c-Fos positive neurons in the bilateral ACC, mPFC and MD (P<0.05). The total distance traveled of OF test remained unchanged. Compared with the model group, 1) EA at ST36, GV20, and HT7 significantly increased the PWTs (P<0.01, P<0.05), 2) EA at ST36 significantly increased the number of entries into the open arms, time spent in the open arms in the EPM test, and increased the number of entries, time spent, and distance traveled in the central area in the OF test (P<0.01 P<0.05), 3) EA at GV20 significantly increased the time spent in the open arms in the EPM test (P<0.01), 4) EA at HT7 obviously increased the time spent in the open arms of the EPM test and increased the time spent in the central area of the OF test (P<0.01), and 5) EA at ST36 significantly decreased the expression of c-Fos in the bilateral ACC, contralateral mPFC, bilateral PVT, and bilateral MD (P<0.05). Compared with the EA ST36 group, the EA ST36 + naloxone group showed a significant decrease in the PWTs (P<0.01), number of entries into the open arms, total distance traveled and time spent in the open arms in the EPM test (P<0.05), and in the number of entries into the central area and time spent in the central area in the OF test (P<0.01). The expression levels of c-Fos in the bilateral ACC and ipsilateral MD were strikingly higher in the EA ST36 + naloxone group than those in the EA ST36 group (P<0.05), suggesting a disappearance of the suppressive effect of EA after administration of naloxone.

EA at ST36 has a better analgesic and emotional relief effects on mice with comorbid pain and emotion compared to EA at GV20 and HT7. The underlying mechanism may involve the inhibition of neuronal excitability in the ACC, mPFC, MD and PVT. Additionally, the emotional relief effect of EA at ST36 is dependent on its analgesic efficacy, which may be related to the inhibition of neuronal excitability in the ACC and MD.