This study examined how repeated electroacupuncture (EA) produces a cumulative analgesic effect, testing whether targeted regulation of the AMPK signaling pathway in the lumbar spinal dorsal horn mediates that effect. The work used a mouse model of inflammatory pain induced by complete Freund's adjuvant (CFA).
Mice were randomized into control, model (CFA), single EA, repeated EA, and sham EA groups; a separate set included vehicle + EA and inhibitor (Compound C) + EA groups. Each group contained eight mice. The inflammatory pain model was created by CFA injection. Repeated EA, vehicle + EA and inhibitor + EA groups received EA at left Zusanli (ST36) and Shangjuxu (ST37) with parameters set at 2 Hz frequency, 1 mA current, 30 minutes per session, once daily for six consecutive days. The single EA group received only one EA session on the second day after modeling. The sham EA group underwent superficial subcutaneous puncture without electrical stimulation.
Compound C (an AMPK inhibitor) was administered intraperitoneally at 20 mg/kg 30 minutes before daily EA in the inhibitor + EA group.
Behavioral endpoints included mechanical pain threshold and thermal pain latency to evaluate nociceptive behavior. Molecular analyses of the lumbar spinal dorsal horn used Western blot to quantify phosphorylated AMPK (p-AMPK), total AMPK and c-Fos protein levels, and immunofluorescence staining to count c-Fos–positive neurons.
CFA injection produced the expected inflammatory pain phenotype: both mechanical pain threshold and thermal pain latency were significantly decreased in the model group compared with controls (P < 0.05). Single EA produced a short-term analgesic effect, with significantly increased mechanical thresholds and thermal latencies at 1, 2 and 4 hours after treatment compared with the model group (P < 0.05). Repeated EA produced sustained increases in pain thresholds after interventions (P < 0.05).
In the vehicle + EA group, pain thresholds on intervention days 2–6 were higher than in the model group (P < 0.05), consistent with a cumulative analgesic effect across repeated sessions. Pre-treatment with the AMPK inhibitor (Compound C) reduced the analgesic benefit: the inhibitor + EA group showed significantly lower mechanical and thermal thresholds on days 2–6 than the vehicle + EA group (P < 0.05).
Repeated EA increased spinal AMPK activation: the ratio of p-AMPK to total AMPK in the lumbar spinal dorsal horn was significantly higher in the repeated EA group than in control and model groups (P < 0.05). The sham EA group displayed a lower p-AMPK/AMPK ratio compared with the EA-treated groups (P < 0.05), indicating that needle insertion without electrical stimulation did not elicit the same AMPK response.
CFA increased neuronal activity markers in the dorsal horn: c-Fos protein expression and the number of c-Fos–positive neurons were significantly elevated in the model group versus control (P < 0.05). Repeated EA (vehicle + EA) reduced both c-Fos expression and c-Fos–positive neuron counts in the lumbar dorsal horn (P < 0.05), consistent with decreased neuronal activation correlating with behavioral analgesia.
Pharmacologic inhibition of AMPK with Compound C reversed key EA effects. Inhibitor + EA mice showed a decline in the pain thresholds achieved by vehicle + EA across days 2–6 (P < 0.05), indicating that blocking AMPK activity attenuated the cumulative analgesic benefit of repeated EA. Similarly, Compound C reversed the EA-associated reductions in c-Fos protein expression and the number of c-Fos–positive neurons in the lumbar dorsal horn (P < 0.05).
These parallel behavioral and molecular reversals support a role for sustained AMPK activation in mediating the cumulative analgesic effect of repeated electroacupuncture, with downstream modulation of neuronal activity as reflected by c-Fos.
The authors conclude that repeated EA effectively alleviated CFA-induced inflammatory pain in mice, and that the cumulative analgesic effect may be mediated by persistent activation of AMPK in the lumbar spinal dorsal horn. Reduced expression of the neuronal activity marker c-Fos accompanied the behavioral improvements, and pharmacologic blockade of AMPK with Compound C attenuated both analgesia and c-Fos reduction.
These findings position spinal AMPK activation as a candidate molecular mediator of EA-induced cumulative analgesia in this inflammatory pain model. If replicated and extended, the data suggest that modulation of AMPK signaling could be a targetable mechanism for enhancing or predicting analgesic responses to repeated EA treatments.
The source material for this summary is the article abstract. Specific numerical data for behavioral measures, full Western blot quantification values, timing of molecular assays relative to the last EA session, and broader mechanistic pathway details were not reported in the abstract and therefore are not included here. The abstract also does not report adverse events, long-term follow-up beyond six days, nor translational data to clinical populations; those details would require the full text.