Changes in short-chain fatty acids affect brain development in mice with early life antibiotic-induced dysbacteriosis

Abstract

Background: Early enteral nutrition and the gut microbiota profoundly influence neonatal brain development, with short-chain fatty acids (SCFAs) from the microbiota playing a pivotal role. Understanding the relationship between dysbiosis, SCFAs, and brain development is crucial. In this study, we investigated the impact of antibiotics on the concentration of SCFAs in neonatal feces. Additionally, we developed a model of gut dysbiosis in neonatal mice to examine the potential relationship between this imbalance, SCFAs production, and brain function development.

Methods: We measured the SCFAs content in the feces of two groups of neonates, categorized based on whether antibiotics were used, and conducted the Neonatal Behavioral Neurological Assessment (NBNA) test on all neonates. Then we evaluated fecal SCFAs levels in neonates and neonatal mice post-antibiotic treatment using liquid chromatography-mass spectrometry (LC-MS) analysis. Morris water maze (MWM) tests assessed behavioral performance, and western blot analysis examined brain tissue-related proteins-neuron-specific enolase (NSE), ionized calcium binding adaptor molecule-1 (IBA1), and myelin basic proteins (MBP).

Results: The use of antibiotics did not affect the NBNA scores of the two groups of neonates, but it did reduce the SCFAs content in their feces. Antibiotic administration induced gut dysbiosis in mice, resulting in decreased IBA1 and MBP expression. Interventions to restore gut microbiota ameliorated these effects. Mice with dysbiosis displayed cognitive deficits in the MWM test. SCFAs levels decreased during dysbiosis, and increased upon microbiota recovery.

Conclusions: Neonatal dysbiosis affects the microbiota-gut-brain axis, impairing cognitive function and nervous system development. Reduced SCFAs may contribute significantly to these alterations.

Keywords: Gastrointestinal microbiome; ionized calcium binding adaptor molecule-1 (IBA1); myelin basic proteins (MBP); neuron-specific enolase (NSE); short-chain fatty acid (SCFA).

Figure 1

Study design. C57BL/6J mice were oral antibiotic by gavage or vehicle (water) from days 1 to 7. From day 8 to day 21, mice were given Bifidobacterium suspension, fecal bacteria suspension or vehicle (water). Con group: mice oral vehicle (water) by gavage twice daily for 21 days; bif group: mice oral antibiotic by gavage twice daily for 7 days and Bifidobacterium suspension for 14 days; fmt group: mice oral antibiotic by gavage twice daily for 7 days and fecal microbiota suspension for 14 days; neg group: mice oral antibiotic by gavage twice daily for 7 days and vehicle (water) for 14 days. MWM, Morris water maze; SCFA, short-chain fatty acid.

Figure 2

After one week of antibiotic treatment in mice, gut dysbiosis occurred. Subsequent administration of Lactobacillus via gavage and fecal microbiota transplantation for two weeks resulted in varying degrees of gut microbiota restoration. (A) α-diversity; (B) β-diversity (day 7); (C) β-diversity (day 21); (D) phylum abundance; (E) family abundance. Con group: mice oral vehicle (water) by gavage twice daily for 21 days; anti group: mice oral antibiotic by gavage twice daily for 7 days, including the con group and the neg group; bif group: mice oral antibiotic by gavage twice daily for 7 days and Bifidobacterium suspension for 14 days; fmt group: mice oral antibiotic by gavage twice daily for 7 days and fecal microbiota suspension for 14 days; neg group: mice oral antibiotic by gavage twice daily for 7 days and vehicle (water) for 14 days.

Figure 3

SCFAs concentrations in fecal pellets measured by mass spectrometry indicate that gut microbiota imbalance affects SCFA production, with butyrate being the most severely impacted and least readily restored. (A) The concentration of AA in the feces of different groups of mice at various time points; (B) the concentration of PA in the feces of different groups of mice at various time points; (C) the concentration of BA in the feces of different groups of mice at various time points. The blanks indicated that the concentrations of PA and BA were too low to be detected on day 1 and day 7. Con group: mice oral vehicle (water) by gavage twice daily for 21 days; bif group: mice oral antibiotic by gavage twice daily for 7 days and Bifidobacterium suspension for 14 days; fmt group: mice oral antibiotic by gavage twice daily for 7 days and fecal microbiota suspension for 14 days; neg group: mice oral antibiotic by gavage twice daily for 7 days and vehicle (water) for 14 days. SCFA, short-chain fatty acid; AA, acetic acid; PA, propionic acid; BA, butyric acid.

Figure 4

Gut dysbiosis mice exhibit learning disability in the MWM, which was remitted by gut microbiota rebuild intervention. Spatial learning was assessed as a function of training day with respect to the following parameters: (A) escape latency and (B) percentage of time spent in the target quadrant. Repeated-measures ANOVA analyzed data of MWM. (A) a: P<0.001, con versus bif or fmt; b: P<0.001, bif or fmt versus neg; c: P<0.001, neg versus con. (B) a: P<0.001, con versus bif; b: P=0.001, bif versus fmt; c: P<0.001, con versus fmt; d: P<0.001, fmt versus neg. Spatial memory is evaluated as a function of detection tests on the following parameters: (C) the percentage of the time spent in the target quadrant and (D) platform crossover number. Repeated-measures ANOVA analyzed data of MWM. Con group: mice oral vehicle (water) by gavage twice daily for 21 days; bif group: mice oral antibiotic by gavage twice daily for 7 days and Bifidobacterium suspension for 14 days; fmt group: mice oral antibiotic by gavage twice daily for 7 days and fecal microbiota suspension for 14 days; neg group: mice oral antibiotic by gavage twice daily for 7 days and vehicle (water) for 14 days. MWM, Morris water maze; ANOVA, analysis of variance.

Figure 5

Expression level of NSE, IBA1 and MBP in vivo experiment. NSE expression showed no significant differences among groups, while IBA1 and MBP were affected by gut microbiota dysbiosis. Con group: mice oral vehicle (water) by gavage twice daily for 21 days; bif group: mice oral antibiotic by gavage twice daily for 7 days and Bifidobacterium suspension for 14 days; fmt group: mice oral antibiotic by gavage twice daily for 7 days and fecal microbiota suspension for 14 days; neg group: mice oral antibiotic by gavage twice daily for 7 days and vehicle (water) for 14 days. MBP, myelin basic proteins; IBA1, ionized calcium binding adaptor molecule-1; NSE, neuron-specific enolase.

Figure 6

Photomicrographs of immunohistochemical markers showing NSE positivity, IBA1 positivity, and MBP positivity (20×). NSE expression showed no significant differences among groups, while IBA1 and MBP were affected by gut microbiota dysbiosis. Con group: mice oral vehicle (water) by gavage twice daily for 21 days; bif group: mice oral antibiotic by gavage twice daily for 7 days and Bifidobacterium suspension for 14 days; fmt group: mice oral antibiotic by gavage twice daily for 7 days and fecal microbiota suspension for 14 days; neg group: mice oral antibiotic by gavage twice daily for 7 days and vehicle (water) for 14 days. IBA1, Ionized calcium binding adaptor molecule-1; MBP, myelin basic proteins; NSE, neuron-specific enolase; ns, none significant.