Chronic pain is a major clinical problem with limited treatment options. Previous studies have demonstrated that activation of adenosine monophosphate-activated protein kinase (AMPK) can attenuate neuropathic pain. Inflammation/immune response at the site of complete Freund’s adjuvant (CFA) injection is known to be a critical trigger of the pathological changes that produce inflammatory pain. However, whether activation of AMPK produces an analgesic effect through inhibiting the proinflammatory cytokines, including interleukin-1β (IL-1β), in inflammatory pain remains unknown. Inflammatory pain was induced in mice injected with CFA. The effects of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside, an AMPK activator), Compound C (an AMPK inhibitor), and IL-1ra (an IL-1 receptor antagonist) were tested at day 4 after CFA injection. Inflammatory pain was assessed with von Frey filaments and hot plate. Immunoblotting, hematoxylin and eosin (H&E) staining, and immunofluorescence were used to assess inflammation-induced biochemical changes. The AMPK activator AICAR produced an analgesic effect and inhibited the level of proinflammatory cytokine IL-1β in the inflamed skin in mice. Moreover, activation of AMPK suppressed CFA-induced NF-κB p65 translocation from the cytosol to the nucleus in activated macrophages (CD68+ and CX3CR1+) of inflamed skin tissues. Subcutaneous injection of IL-1ra attenuated CFA-induced inflammatory pain. The AMPK inhibitor Compound C and AMPKα shRNA reversed the analgesic effect of AICAR and the effects of AICAR on IL-1β and NF-κB activation in inflamed skin tissues. Our study provides new information that AMPK activation produces the analgesic effect by inhibiting NF-κB activation and reducing the expression of IL-1β in inflammatory pain.

This protocol establishes a comprehensive experimental framework for investigating cortical circuit dynamics during EA (electroacupuncture) stimulation. The methodology integrates three key components: chronic cranial window implantation in Thy1-GCaMP6f transgenic mice, standardized EA stimulation at the ST36 acupoint using 2/100 Hz biphasic pulses, and high-resolution two-photon calcium imaging. This integrated approach enables real-time visualization of neural ensemble activity in the primary somatosensory (S1) cortex with millisecond temporal precision, capturing previously inaccessible details of network-level responses to neuromodulation. The technique successfully reveals distinct layer-specific activation patterns and longitudinal plasticity changes, providing critical insights into the cortical mechanisms underlying the effects of EA. By overcoming the fundamental spatiotemporal limitations inherent in conventional fMRI and electrophysiological techniques, this high-resolution platform offers unprecedented analytical capabilities for mapping dynamic neural circuits. The protocol's robust design and reproducible outcomes make it particularly valuable for optimizing targeted neuromodulation therapies and advancing our understanding of circuit-level plasticity in response to peripheral stimulation.

This protocol establishes a comprehensive experimental framework for investigating cortical circuit dynamics during EA (electroacupuncture) stimulation. The methodology integrates three key components: chronic cranial window implantation in Thy1-GCaMP6f transgenic mice, standardized EA stimulation at the ST36 acupoint using 2/100 Hz biphasic pulses, and high-resolution two-photon calcium imaging. This integrated approach enables real-time visualization of neural ensemble activity in the primary somatosensory (S1) cortex with millisecond temporal precision, capturing previously inaccessible details of network-level responses to neuromodulation. The technique successfully reveals distinct layer-specific activation patterns and longitudinal plasticity changes, providing critical insights into the cortical mechanisms underlying the effects of EA. By overcoming the fundamental spatiotemporal limitations inherent in conventional fMRI and electrophysiological techniques, this high-resolution platform offers unprecedented analytical capabilities for mapping dynamic neural circuits. The protocol's robust design and reproducible outcomes make it particularly valuable for optimizing targeted neuromodulation therapies and advancing our understanding of circuit-level plasticity in response to peripheral stimulation.