Background: Systemic inflammation and insomnia often co-occur in patients with depression. However, there is no suitable animal model to investigate the relationship between inflammation, sleep deprivation (SD), and depression. Methods: To model interactions between insomnia, inflammation, and depression, we developed a novel "two-hit" rodent model of depressive-like behaviors using continuous SD followed by daily lipopolysaccharide (LPS) treatment. Control groups received SD, LPS, or sterile phosphate-buffered salinealone. The model's validity was assessed at the cellular and molecular levels, with fluoxetine rescue applied to confirm model validity. Results: The model group demonstrated significant depressive-like behaviors that were rescued by fluoxetine treatment. Transcriptomic analysis revealed alterations in neuroinflammation and synaptic plasticity pathways within the hippocampus and prefrontal cortex (PFC) of model rats. Western blotting validated alterations in key protein markers related to both processes, and immunofluorescence confirmed microglia and astrocyte activation, indicative of neuroinflammation. Additionally, transmission electron microscopy and Golgi-Cox staining revealed reduced synapse and dendritic spine density in the model group. Fluoxetine treatment reversed these structural changes. Sixteen genes associated with neuroinflammation and synaptic function were validated in human genetic studies by transcriptome-wide association analysis. Conclusion: This reliable two-hit model will be useful for investigating the roles of insomnia and inflammation in depression.

The authors have declared no competing interest.

This work was supported by grants from the National Natural Science Foundation of China [grant numbers: U21A20364], the National Key Research and Development Program of China [grant number: 2018YFC1314600] and the Second Medical Leading Talent in Hubei Province.

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