Impact of prolonged continuous exposure to stress on immune function and gut microbiome in a perpetual avoidance of water on a wheel mouse model

Response to stress involves a complex interplay between the immune system and the gut microbiome, yet studies on long-term continuous stress remain scarce due to the lack of suitable models. In this study, we employed our previously developed Perpetual Avoidance of Water on a Wheel (PAWW) model to investigate the effects of prolonged stress on immune-related cytokine expression and gut microbiome composition in mice. We demonstrated that PAWW stress can be sustained for six weeks without signs of behavioral or physiological adaptation. Circadian locomotor rhythms remained disrupted throughout the exposure period, accompanied by elevated plasma norepinephrine and epinephrine. Quantitative real-time PCR revealed significant upregulation of inflammation-related cytokine genes, including TNF-α in the brain and IL-6 and IL-1β in intestinal Peyer’s patches (PPs). Microbiome profiling by 16S rRNA gene sequencing showed that stressed mice underwent more pronounced compositional changes than controls, particularly in members of the Lachnospiraceae family. These findings indicate that the PAWW model provides a robust platform for investigating the effects of chronic stress on the brain–immune–gut axis. Prolonged PAWW stress was associated with both inflammatory immune responses and microbiome alterations, highlighting a potential role for PPs in mediating intestinal immune regulation under stress conditions.