Abstract Body: Introduction
Stroke is a leading cause of death and disability worldwide. Among the risk factors for stroke and cardiovascular disease (CVD), gut microbiota-derived trimethylamine N-oxide (TMAO) has emerged as one of key biomarker. Elevated TMAO levels are associated with vascular dysfunction by promoting atherosclerosis, inflammation, oxidative stress, platelet hyperreactivity, and increasing CVD risk. Effective strategies to lower TMAO remain limited. Among the possible strategies, probiotics therapies targeting TMAO offer a novel approach with potential.

Hypothesis
We hypothesized that BM109, a newly developed probiotic, could improve stroke outcomes by reducing TMAO-mediated damage in a transient middle cerebral artery occlusion (tMCAO) model under high-choline diet.

Methods
Adult male Wistar rats (8 weeks old) were randomly assigned to eight groups (n=16/group): Chow-Control-Sham, Chow-BM109-Sham, Chow-Control-tMCAO, Chow-BM109-tMCAO, Choline-Control-Sham, Choline-BM109-Sham, Choline-Control-tMCAO, and Choline-BM109-tMCAO. Rats were fed a chow or 1.2% choline diet for 3 weeks. BM109 (5×10¹⁰ CFU/mL/day) or control (15% glycerol) was orally administered during the final week. Sham groups had no occlusion, while tMCAO groups underwent 90 minutes of middle cerebral artery occlusion and reperfusion. Infarct size was measured by TTC staining, and neurological deficits were assessed using blind evaluations of Garcia, Longa, and mNSS tests.

Results
In tMCAO-induced groups, high-choline diets significantly increased infarct size compared to chow diets group (Choline-Control: 30.01±4.67% vs. Chow-Control: 20.35±4.42%, p<0.0001). BM109 significantly reduced the infarct size, particularly under high-choline conditions (Choline-BM109: 12.59±3.40% vs. Choline-Control: 30.01±4.67%, p<0.0001) and improving neurological outcomes (mNSS: Choline-BM109: 10.31±2.73 vs. Choline-Control: 13.13±1.09, p<0.01).

Conclusion
We found that high-choline diets exacerbate stroke severity by increasing gut microbiota-derived TMAO levels. BM109 significantly reduced infarct size and improved neurological deficits, particularly under high-choline conditions. These findings suggest that BM109 may be a promising therapeutic strategy for stroke and other TMAO-associated cardiovascular diseases.