Short-Chain Fatty Acids Analysis in Brain by GC/MS to Determine the Effect of Bioactive Food in a Mouse Model of Alzheimer’s Disease

Importance of the topic

The interaction between gut microbiota and brain function has emerged as a key factor in Alzheimer’s disease (AD) pathology. Short-chain fatty acids (SCFAs) such as propionic acid, produced by gut bacteria, may influence neuroinflammation, synaptic function and cognitive performance. Accurate measurement of SCFAs in brain tissue is essential to understand the gut-brain axis and to evaluate nutritional interventions aimed at modulating disease progression.

Objectives and overview of the study

This study aimed to quantify propionic acid levels in the prefrontal cortex of a transgenic mouse model of AD and to assess the impact of a long-term bioactive food (BF) diet on SCFA concentrations, neuropathological markers and cognitive outcomes. Specific goals included:

• Comparing propionate levels in TG (3×Tg-AD) and wild-type mice.
• Evaluating the effect of BF ingestion on brain SCFA content and receptor expression.
• Determining whether dietary intervention reduces amyloid and tau pathology and improves working memory.

Methodology

Female 3×Tg-AD mice (homozygous APPSWE, PS1M146V, TauP301L) and WT B6129SF1/J controls were maintained on either a standard diet (AIN) or BF for seven months from age two to nine months. Cognitive performance was assessed using a T-maze spontaneous alternation test. Brain samples were collected for immunohistochemistry, immunofluorescence and Western blot analyses of inflammatory and synaptic markers. Fecal microbiota profiling complemented biochemical assays of SCFAs in both feces and cortex.

Used instrumentation

• GC/MS system: Agilent Intuvo 9000 gas chromatograph coupled to Agilent 5977B mass selective detector.
• Injection: Thermal separation probe for direct introduction of weighed brain samples; split injection (20:1), injection volume 0.4 µL.
• Column: DB-WAX UI (30 m × 0.25 mm × 0.5 µm).
• Carrier gas: Helium at 1 mL/min; oven program: 60 °C (0.05 min), ramp to 250 °C at 900 °C/min, hold 3 min.
• Data analysis: Agilent MassHunter Quantitative software.

Main results and discussion

• Propionic acid in prefrontal cortex was significantly higher in TG-AIN mice than in WT, and BF ingestion reduced these levels to near-control values (p < 0.05).
• Expression of the propionate receptor FFAR3/GPR41 was elevated in TG-AIN cortex and normalized by BF diet.
• TG mice on standard diet exhibited impaired T-maze performance, while BF-fed TG mice showed restored working memory.
• BF intake attenuated amyloid-β aggregation, tau hyperphosphorylation, neuroinflammation and synaptic/metabolic alterations associated with AD pathology.
• Body weight was higher in TG mice but unaffected by BF; no changes in food intake or weight gain were observed.

Benefits and practical applications of the method

Direct GC/MS quantification of SCFAs in brain tissue using a thermal separation probe enables rapid, sensitive analysis without extensive sample preparation. This approach facilitates exploration of dietary and microbial influences on central metabolite levels and supports development of nutritional interventions for neurodegenerative diseases.

Future trends and potential applications

• Expansion to other SCFAs and brain regions to map the full metabolic signature of the gut-brain axis.
• Integration with metabolomic and imaging techniques to correlate chemical changes with functional alterations in vivo.
• Translational studies evaluating BF formulations or probiotics in early-stage AD patients.
• Development of high-throughput, automated platforms for routine SCFA monitoring in preclinical and clinical research.

Conclusion

Long-term ingestion of bioactive food in a female 3×Tg-AD mouse model modulates gut microbiota, decreases pro-inflammatory propionate levels in the brain, reduces hallmark AD pathology and improves cognitive performance. The described GC/MS method offers a reliable tool for investigating SCFA dynamics in neurodegenerative disease research and for guiding dietary intervention strategies.

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