Cecal microbiota and ipsilateral hippocampal metabolic differences at day 7 after permanent middle cerebral artery occlusion in rats: an exploratory multi-omics study

Abstract Background Gut microbial alterations and brain metabolic disturbances have both been reported after cerebral ischemia, but their relationship with region-specific metabolic changes in the hippocampus remains unclear. This study characterized gut microbial and ipsilateral hippocampal metabolic alterations and evaluated hippocampal inflammatory and inflammasome-associated changes during the early subacute phase after permanent middle cerebral artery occlusion (MCAO). Methods Male Sprague-Dawley rats were assigned to Naïve or permanent MCAO groups. On day 7, paired cecal content and ipsilateral hippocampal samples collected from the same rats were analyzed by 16 S rRNA gene sequencing and untargeted UPLC-MS/MS metabolomics ( n = 8/group). Spearman correlation analysis was performed using matched microbiota and metabolomic profiles. In separate animal subsets, cerebral perfusion, infarction, hippocampal neuronal injury, inflammatory and eNOS-related markers, astrocytic reactivity, and inflammasome-associated proteins were assessed using laser speckle contrast imaging, TTC staining, histology, immunohistochemistry, immunofluorescence, and ELISA. GSE22255 was examined as contextual evidence of longer-term peripheral inflammatory gene expression after human ischemic stroke. Results Permanent MCAO was accompanied by reduced microbial richness and diversity and a distinct cecal microbial composition. The relative abundances of Escherichia-Shigella , Erysipelotrichaceae_UCG-003 , and Parabacteroides were higher, whereas that of Bifidobacterium was lower, in MCAO rats. Hippocampal metabolomics revealed a lipid-centered metabolic phenotype characterized by increased signals putatively annotated as lysophosphatidylcholines, L-palmitoylcarnitine, butyrylcarnitine, and taurohyocholate, together with a decreased signal putatively annotated as homocarnosine. Pooled cross-omic analysis identified significant genus–metabolite associations, and the Erysipelotrichaceae_UCG-003 –taurohyocholate association remained significant after adjustment for group membership. MCAO rats also exhibited reduced cerebral perfusion, cerebral infarction, hippocampal neuronal injury, increased TNF-α immunoreactivity and GFAP immunoreactivity, reduced eNOS immunoreactivity, and higher hippocampal levels of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18. Analysis of GSE22255 showed higher LBP, NLRP3, and IL1B expression in peripheral blood mononuclear cells collected at least 6 months after stroke. Conclusions At day 7 after permanent MCAO, cecal microbiota remodeling and a lipid-related metabolic phenotype in the ipsilateral hippocampus were observed, with inflammatory and inflammasome-associated changes identified across separate experimental subsets. Paired cross-omic associations provide hypotheses for subsequent mechanistic investigation but do not establish a causal gut–brain relationship.