Study of the mechanism of electroacupuncture for alleviating chronic hyperalgesia in mice through modulation of spinal dorsal horn microglia

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.