Analysis of the mouse brain metabolome following the disruption of the gut-brain axis

Importance of the Topic

The gut-brain axis represents a critical communication network linking the intestinal microbiome with central nervous system metabolism. Disruptions to this axis contribute to neurological disorders, making comprehensive brain metabolome profiling essential for understanding host–microbe interactions and guiding therapeutic interventions.

Objectives and Study Overview

This study investigated how Clostridioides difficile infection (CDI) and subsequent metronidazole treatment alter the mouse brain metabolome. Using an untargeted high-resolution LC-MS/MS workflow, the authors compared metabolite profiles in control mice, infected mice, and infected mice receiving antibiotic therapy to identify significant biochemical changes associated with gut-brain axis perturbation.

Instrumentation Used

• Shimadzu LCMS-9030 QTOF mass spectrometer with ESI positive/negative modes
• Acquity C18 BEH column (2.1×100 mm, 1.7 µm) for reverse-phase separation
• Shim-pack Velox HILIC column (2.1×100 mm, 2.7 µm) for polar metabolite analysis

Methodology and Data Processing

Brain tissues were extracted using methanol:isopropanol:water and MTBE-based protocols. Reverse-phase LC-MS/MS (35 min cycle) targeted lipids and mid-polarity compounds, while HILIC LC-MS/MS (18 min cycle) focused on early-eluting polar metabolites. MS-Dial aligned chromatographic features; signals present in fewer than 80 % of QCs or with RSD > 20 % were excluded. Statistically significant features were determined by ANOVA (p < 0.05, FDR corrected) in MetaboAnalyst. Metabolite annotation reached MSI level 1 via an in-house MS/MS library and level 2 through MassBank, mzCloud, and LipidMaps.

Results and Discussion

ANOVA highlighted over 20 metabolites showing significant changes across groups. Key findings:

• Elevated nucleosides (cytidine, cytosine, inosine, uridine, 5′-methylthioadenosine) in infected mice, further increased by metronidazole.
• Reduced free fatty acids (arachidonic acid, docosapentaenoic acid), sphingomyelins (SM 34:0, SM 34:1) and malate following infection, partially restored by antibiotic treatment.
• Carnitine derivatives (acetylcarnitine, arachidonoylcarnitine) and carboxylic acids displayed opposite trends, with infection elevating levels and treatment reducing them.
• HILIC analysis improved confidence for early-eluting compounds such as acetylcarnitine and cytidine by matching product ion spectra to authentic standards.

Benefits and Practical Applications

This workflow demonstrates how combining reverse-phase and HILIC LC-MS/MS can comprehensively profile brain metabolites impacted by gut microbiome perturbations. Insights into metabolite shifts support biomarker discovery for neuroinflammation, antibiotic impact assessment, and mechanistic studies of gut-brain disorders.

Future Trends and Opportunities

Advances in spatial metabolomics and integration with metagenomics will enable mapping of microbial-host interactions in situ. Expanded high-confidence MS/MS libraries and AI-driven annotation tools promise faster, more accurate metabolome coverage. Personalized metabolomic profiling may guide targeted therapeutics for gut-brain axis disorders.

Conclusion

This study reveals that CDI and metronidazole exert additive effects on the mouse brain metabolome, altering nucleosides, lipids, and energy metabolites. The dual-mode LC-MS/MS strategy, supported by rigorous data processing and MS/MS confirmation, provides a robust platform for investigating gut-brain metabolic crosstalk.