Optogenetic stimulation of the “Zusanli” acupoint alleviates inflammatory pain through active Wnt/β-Catenin and MAPK signaling pathway in rats

Inflammatory pain, an important form of common pain, negatively influences the quality of life. Pathway-selective optogenetic control is a popular tool in neuronal function research; however, attempts to modulate rodent behavior using pathway-selective optogenetics remain unverified. We developed a methodology for pathway-selective optogenetics in rats, focusing on the delivery of recombinant adeno-associated virus (rAAV) containing channelrhodopsin-2 (ChR2) injected at the "Zusanli" acupoints to the dorsal root ganglia (DRG) and toes, which is a part of the complex neuron network. Optogenetic stimulation of gamma-aminobutyric acid (GABA) projections to the "Zusanli" acupoints delivered several rAAV9-GAD1-ChR2-mcherry particles to the spinal cord horn (SCDH) and DRG and few rAAV9-GAD1-ChR2-mcherry particles to toes, thereby evoking an analgesic effect in complete Freund's adjuvant (CFA) rats similar to those of acupuncture. Furthermore, blue light stimulation (LED) and electroacupuncture (EA) reduced inflammatory pain in CFA rats, as evidenced by the changes in the paw edema, paw withdrawal latency (PWL), and paw withdrawal threshold (PWT) values. Further exploration of the mechanisms underlying this phenomenon revealed that optogenetic stimulation upregulates the decarboxylase-65 (GAD65) and decarboxylase-67 (GAD67) protein levels and downregulates the levels of GABA transporter-1 (GAT1) and GABA transporter-3 (GAT3) to alleviate inflammatory pain. The activation of the p38 mitogen-activated protein kinases (MAPK) and Wnt signal transduction pathways decreased the release of interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis factor-α (TNFα). These findings indicate that optogenetic stimulation of the "Zusanli" acupoint alleviated inflammatory pain.