Time‐Dependent Multisystem Effects of Small Independent Space ( SIS ) Exposure in Mice: Integrative Analysis of Behavior, Neuroendocrine, Gut Microbiota, and Hippocampal Function

Exposure to small independent space (SIS) causes stress-related behavioral and neural abnormalities, but the time-dependent mechanisms and gut microbiota-hippocampus interactions remain unclear. To investigate the time-dependent effects of acute and chronic restraint stress (CRS) on mouse behavior, neuroendocrine function, gut microbiota, and hippocampal activity. Behavioral assessments were conducted using the open field test and tail suspension test. Meanwhile, enzyme-linked immunosorbent assay (ELISA) was applied to measure neurotransmitters, hypothalamic-pituitary-adrenal (HPA) axis markers, and inflammatory factors; 16S rRNA sequencing was used for gut microbiota analysis; liquid chromatography-tandem mass spectrometry (LC-MS/MS) for metabolite identification; and immunofluorescence staining for hippocampal structure observation. Acute SIS induced depression-like behaviors. Chronic SIS showed peak despair at 4 days (d), followed by persistent depression at 8 d. Norepinephrine (NE) increased while 5-hydroxytryptamine (5-HT) decreased; NE depletion at 4 d coincided with reduced hippocampal glucocorticoid receptor (GR) expression. Both acute (2 h) and chronic (4 d) SIS reduced gut microbiota diversity and disrupted tyrosine metabolism. Hippocampal tests showed neuronal compensation at 6 h post-acute stress and neuronal apoptosis with glial dysfunction after 4 d of chronic stress. SIS damages via the "gut microbiota-tyrosine metabolism-hippocampus axis," with 4 d of chronic exposure as a critical decompensation point, offering a time-specific intervention target.