IL-1 Mediates Chronic Stress-Induced Hyperalgesia Accompanied by Microglia and Astroglia Morphological Changes in Pain-Related Brain Regions in Mice

**Background:** Chronic primary pain conditions such as fibromyalgia and complex regional pain syndrome are poorly understood and lack effective therapies. These conditions are more prevalent in women and are strongly associated with stress. Interleukin-1 (IL-1) is a pro-inflammatory cytokine known to be involved in stress responses and inflammatory pain, but its role in stress-induced pain syndromes had not been investigated. This study aimed to determine whether IL-1 mediates chronic stress-induced hyperalgesia and associated glial changes in pain-related brain regions.

**Methods:** Female and male C57Bl/6J wild-type (WT) and IL-1αβ-deficient (knockout; KO) mice were subjected to chronic restraint stress (CRS) — 6 hours of immobilization per day for 4 weeks. Mechanonociceptive thresholds were measured weekly using dynamic plantar aesthesiometry (DPA; max force 10 g, ramp 4 s). Cold tolerance was assessed by measuring hind paw withdrawal latency from 0°C water (cut-off 180 s). Behavioral tests for anxiety (light-dark box, open field test) and depression-like behavior (forced swim test, tail suspension test) were performed after 2 and 4 weeks. Relative thymus and adrenal gland weights were measured as indicators of endocrine stress responses. Immunohistochemical analysis of microglia (IBA1) and astrocytes (GFAP) was performed in four pain-related brain regions: central nucleus of the amygdala (CeA), primary somatosensory cortex hind limb representation (S1HL), hippocampus CA3, and periaqueductal gray (PAG). Integrated density, cell number, and activation scores (1–5 scale) were quantified using ImageJ.

**Key Results:** CRS induced 15–20% mechanical hyperalgesia after 2 weeks in WT mice of both sexes. This effect was significantly reduced in female IL-1 KO mice but not in male IL-1 KOs. Cold hypersensitivity (60–70% increased sensitivity) developed in all groups regardless of genotype or sex. Female IL-1 KO mice had significantly higher baseline mechanonociceptive thresholds compared to WT females. After 2 weeks of CRS, IBA1+ integrated density was significantly increased in the CeA, S1HL, hippocampus CA3, and PAG of stressed WT females but not in IL-1 KOs. IBA1+ microglia cell number was significantly increased in the PAG of stressed WT mice compared to KOs. GFAP+ astrocyte activation scores were significantly elevated in the hippocampal CA3 of stressed WT but not KO mice. Relative adrenal gland weights increased and thymus weights decreased after 2 weeks of CRS in WT females, but these differences vanished by week 4, indicating habituation. No significant anxiety- or depression-like behaviors were detected in WT mice after CRS. Interestingly, IL-1 KO mice showed increased locomotor activity and decreased immobility in the forced swim test and tail suspension test after 2 weeks of stress.

**Clinical Implications:** This study provides the first evidence that IL-1 mediates chronic stress-induced mechanical hyperalgesia in female mice, accompanied by microglial and astroglial changes in pain-related brain regions. The sex-specific effect aligns with the higher prevalence of stress-related chronic pain conditions (e.g., fibromyalgia) in women. The findings suggest that IL-1 contributes to central sensitization rather than peripheral sensitization, as mechanical hyperalgesia (centrally mediated) was IL-1-dependent while cold hypersensitivity (peripherally mediated) was not. The authors propose that inhibiting IL-1 receptor 1 (IL-1R1) could represent a potential therapeutic opportunity for chronic primary pain conditions such as fibromyalgia and complex regional pain syndrome. Limitations include the use of global knockout mice from embryonic age, which may result in compensatory changes in other inflammatory mediators.