Sex differences in the fecal microbiome and hippocampal glial morphology following diet and antibiotic treatment

Abstract

Rising obesity rates have become a major public health concern within the United States. Understanding the systemic and neural effects of obesity is crucial in designing preventive and therapeutic measures. In previous studies, administration of a high fat diet has induced significant weight gain for mouse models of obesity. Interestingly, sex differences in high-fat diet-induced weight gain have been observed, with female mice gaining significantly less weight compared to male mice on the same high-fat diet. It has also been observed that consumption of a high-fat diet can increase neurogliosis, but the mechanism by which this occurs is still not fully understood. Recent research has suggested that the gut microbiome may mediate diet-induced glial activation. The current study aimed to (1) analyze changes to the gut microbiome following consumption of a high fat (HF) diet as well as antibiotic treatment, (2) evaluate hippocampal microgliosis and astrogliosis, and (3) identify sex differences within these responses. We administered a low fat (Research Diets D12450 K) or high fat diet (Research Diets D12451) to male and female C57Bl/6 mice for sixteen weeks. Mice received an antibiotic cocktail containing 0.5g/L of vancomycin, 1.0 g/L ampicillin, 1.0 g/L neomycin, and 1.0 g/L metronidazole in their drinking water during the last six weeks of the study and were compared to control mice receiving normal drinking water throughout the study. We observed a significant reduction in gut microbiome diversity for groups that received the antibiotic cocktail, as determined by Illumina next-generation sequencing. Male mice fed the HF diet (± antibiotics) had significantly greater body weights compared to all other groups. And, female mice fed the low fat (LF) diet and administered antibiotics revealed significantly decreased microgliosis and astrogliosis in the hippocampus compared to LF-fed females without antibiotics. Interestingly, male mice fed the LF diet and administered antibiotics revealed significantly increased microgliosis, but decreased astrogliosis, compared to LF-fed males without antibiotics. The observed sex differences in LF-fed mice given antibiotics brings forward questions about sex differences in nutrient metabolism, gut microbiome composition, and response to antibiotics.

Fig 1. Average food consumption and body weight during the 16-week dietary study.

(A) Average food consumption for the entire 16-week study. Different letters indicate groups that are significantly different (p<0.05). (B) HF-fed mice gained significantly more weight than LF-fed mice. Male HF and Male HF Antibiotics gained significantly more weight than all other groups. (C) Male HF Antibiotics reveal the highest body weight; Female LF and Female LF Antibiotics reveal the lowest body weight. Values shown are mean ± SEM.

Fig 2. Fecal microbiome alpha diversity (n = 6/group).

(A) There was a significant effect of treatment (p<0.0001) and a significant interaction effect (sex x treatment; p = 0.0002) for Shannon Diversity. (B) There was a significant effect of treatment (p<0.0001) and a significant effect of sex (p = 0.0021) on Operational Taxonomic Units. (C) There was a significant effect of treatment (p<0.0001) and a significant effect of sex (p = 0.0026) on Faith’s Phylogenetic Diversity.

Fig 3. Principle coordinate plot of weighted UniFrac data (n = 6/group).

The primary vector explains 78.8% of the variation between the groups. The first 3 vectors together exhibit 90.7% of the variation among the groups.

Fig 4. Dual hierarchical dendrogram (n = 6/group).

To provide a visual overview combined with analysis, a dual hierarchal dendrogram was used to display the data for the predominant genera with clustering related to the different groups. Samples with more similar microbial populations were mathematically clustered closer together. The genera (consortium) were used for clustering. The samples with more similar consortium of genera cluster closer together with the length of connecting lines (top of heatmap) related to the similarity, shorter lines between two samples indicate closely-matched microbial consortium. The heatmap represents the relative percentages of each genus. The predominant genera are represented along the right Y-axis. The legend for the heatmap is provided in the upper left corner. The heat map and clustering reveals a significant effect of antibiotic treatment.

Fig 5. Genus-level relative abundances in the fecal microbiome (n = 6/group).

Average relative abundance for the top 14 most abundant genera are displayed for each of the 8 experimental groups.

Fig 6. Microglial analysis (n = 4-5/group).

The results for fractal analysis (A), branch length (B), cell complexity (C), cell shape (D), density (E), and summed endpoints (F) are shown. * p<0.05; *** p<0.001; **** p<0.0001.

Fig 7. Representative micrographs of microglia.

100X images were collected for microglia labeled with the Iba-1 antibody.

Fig 8. Correlations for fractal analysis and Shannon Diversity.

(A) All groups are plotted, including results with low Shannon diversity for antibiotic-treated vs. higher Shannon diversity for mice that received normal drinking water. (B) A positive correlation (Spearman r = 0.5637; p = 0.0203) was determined for antibiotic-treated mice. (C) A negative correlation (Spearman r = -0.5735; p = 0.0223) was determined for normal drinking water mice.

Fig 9. GFAP densitometry (n = 5/group).

Female LF Antibiotics revealed a significant decrease in GFAP densitometry in the hippocampus compared to Female LF. ** p < 0.01. Representative micrographs of images taken at 10X following labeling with a GFAP antibody are presented.