Microglial CX3CR1 signaling mediates stress-induced pain behavior in mice.

Introduction: Chronic primary pain conditions, including fibromyalgia, affect up to 10% of the population, yet their pathophysiology is unexplored and the treatment is insufficient. Chronic stress is a key etiological factor and is known to modulate microglial function, partly via the CX3CR1 fractalkine receptor. Here, we investigated the role of CX3CR1 in a mouse model of stress-induced pain. Methods: Female and male CX3CR1-deficient (KO) and C57Bl/6J wild-type (WT) mice were exposed to chronic restraint stress (CRS) for 2 weeks. Mechanical and cold sensitivity were assessed before and during CRS. Microglia-IBA1 and astrocyte-GFAP activation were analyzed in stress- and pain-related brain regions, and neuron-glia interactions were examined in the somatosensory cortex hindlimb area (S1HL). Pharmacological validation was performed using the CX3CR1 antagonist, AZD8797 in WT mice. Results: In WT animals, CRS induced approximately 20% mechanical and 60-70% cold hyperalgesia. Mechanical pain and cold sensitivity was significantly reduced in stressed CX3CR1 KO mice of both sexes. CRS caused microglia and astrocyte integrated density increases in stress- and pain-related regions in WT but not CX3CR1 KO mice. Microglia coverage of neurons was greater in the S1HL region of KO animals independently of the CRS protocol. Pharmacological blockade of the CX3CR1 abolished CRS-evoked mechanical but not cold hyperalgesia. Discussion: These findings demonstrate that microglial CX3CR1 signaling contributes to chronic stress-induced pain through neuroinflammatory mechanisms and central pain sensitization. Targeting CX3CR1 may represent a promising therapeutic strategy for chronic primary pain conditions such as fibromyalgia.