Disrupted gut microbiota aggravates working memory dysfunction induced by high-altitude exposure in mice

Abstract

Background: The widely accepted microbiome-gut-brain axis (MGBA) hypothesis may be essential for explaining the impact of high-altitude exposure on the human body, especially brain function. However, studies on this topic are limited, and the underlying mechanism remains unclear. Therefore, this study aimed to determine whether high-altitude-induced working memory dysfunction could be exacerbated with gut microbiota disruption.

Methods and results: C57BL/6 mice were randomly divided into three groups: control, high-altitude exposed (HAE), and high-altitude exposed with antibiotic treatment (HAE-A). The HAE and HAE-A groups were exposed to a low-pressure oxygen chamber (60-65 kPa) simulating the altitude of 3,500-4,000 m for 14 days, The air pressure level for the control group was maintained at 94.5 kPa. Antibiotic water (mixed with 0.2 g/L of ciprofloxacin and 1 g/L of metronidazole) was provided to the HAE-A group. Based on the results of the novel object test and P300 in the oddball behavioral paradigm training test, working memory dysfunction was aggravated by antibiotic treatment. We determined the antioxidant capacity in the prefrontal cortex and found a significant negative influence (p < 0.05) of disturbed gut microbiota on the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The same trend was also observed in the apoptosis-related functional protein content and mRNA expression levels in the prefrontal cortex, especially the levels of bcl-2, Bax, and caspase-3. The high-altitude environment and antibiotic treatment substantially affected the richness and diversity of the colonic microbiota and reorganized the composition and structure of the microbial community. S24-7, Lachnospiraceae, and Lactobacillaceae were the three microbial taxa with the most pronounced differences under the stimulation by external factors in this study. In addition, correlation analysis between colonic microbiota and cognitive function in mice demonstrated that Helicobacteraceae may be closely related to behavioral results.

Conclusion: Disrupted gut microbiota could aggravate working memory dysfunction induced by high-altitude exposure in mice, indicating the existence of a link between high-altitude exposure and MGBA.

Keywords: antibiotics; gut microbe; gut-brain axis; high altitude; probiotic.

Figure 1

The schematic diagram of novel object test and oddball paradigm behavior training. (A) Schematic diagram of novel object test. A dark open-field box (40 * 40 * 45 cm) was used. The objects shown to the mice were a smooth pebble, a blue cap of the same size and form, and a orange cap of the same size and form, respectively. (B) Schematic diagram of oddball paradigm behavior training. (C) Schematic presentation of different phases of the Oddball paradigm behavior Training. Animals had to correctly respond to infrequent target (8 Hz) tones while correctly ignoring infrequent standard (6 Hz) tones. The electrical signals of the mice were recorded during the task phase. T referred to Target tone, S referred to Standard tone.

Figure 2

Effects of high altitude and antibiotics on exploration ratio by novel object test. Data are presented with the means ± standard deviation (n = 8). *Difference is significant at the 0.05 level (p < 0.05); **difference is significant at the 0.01 level (p < 0.01); ***difference is significant at the 0.001 level (p < 0.001).

Figure 3

Variation of P300 of the oddball behavioral paradigm training test. (A) The profile of P300 in control, HAE and HAE-A group, respectively; (B) The result of P300 evoked by standard tone stimulation; (C) The result of P300 evoked by target tone stimulation; (D) The result of normalized P300 amplitude by target/standard. Data are presented with the means ± standard deviation (n = 6). *Difference is significant at the 0.05 level (p < 0.05); **difference is significant at the 0.01 level (p < 0.01).

Figure 4

Antioxidant capacity in the prefrontal cortex. Data are presented with the means ± standard deviation (n = 6). NS, not significant (p > 0.05); *difference is significant at the 0.05 level (p < 0.05); **difference is significant at the 0.01 level (p < 0.01); ***difference is significant at the 0.001 level (p < 0.001). (A–F) Activities or contents of T-AOC, SOD, CAT, GSH-Px, MDA, and GSH, respectively. T-AOC, total antioxidation capacity; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; GSH, glutathione.

Figure 5

Apoptosis-related functional protein contents and mRNA expression levels in the prefrontal cortex. Data are presented with the means ± standard deviation (n = 6). Bars with different letters are significantly different on the basis of Duncan’s multiple-range test (p < 0.05). NS, not significant (p > 0.05); *difference is significant at the 0.05 level (p < 0.05); **difference is significant at the 0.01 level (p < 0.01); ***difference is significant at the 0.001 level (p < 0.001). (A,B): mRNA expression levels and protein contents of bcl-2 and Bax; (C–E): mRNA expression levels of Bad, caspase-9 and caspase-3, respectively.

Figure 6

Effects of hypoxia on the structure of colon microbiota in normal mice and antibiotic drinking mice. (A) Intestinal microbial community diversity in each group (Shannon). Meaning: Wilcoxon. (B) Gut microbiome richness (observed species) in colonic luminal samples of each group. Significance: Wilcoxon. (C) Principal Coordinate Analysis (PCoA) of Unweighted UniFrac Distance between groups. (D) Relative abundance at phylum level in each group (%). (E) Relative abundance at each family level (%). (F) Bipartite networks of indicator species analysis. It displays different treatment-specific ASVs in the colonic bacterial communities using indicator species analysis. Circles represent individual bacteria and ASVs that are positively and significantly associated (p < 0.05) with one or more different grouping factors (association(s) given by connecting lines). ASVs are colored according to their Family assignment.

Figure 7

Significant altered bacterial taxa in colonic microbes by different treatments at family level. A–C: S24-7, Lachnospiraceae, and Lactobacillaceae, respectively. Boxplots showing differences in the relative abundance of significantly discriminant taxa between different treatments. Significance between groups was indicated with asterisk (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 8

LEfSe analysis of colon microbiota in mice. Cladogram (A,C) and linear discriminant analysis (LDA) score (B,D) and cladogram (A,C) were generated from LDA effect size. Taxa with LDA values larger than 4 are shown in the figure.

Figure 9

Correlation between microbial community and behavioral test in the colon. (A) Different colors represent positive (red) or negative (blue) correlation of important family-level microbes with behavioral results. Circles and asterisks represent different levels of importance. (B–D) The different results represent the importance of having three species at the family level in behavioral performance. Red circle: control group. Blue circle: HAE group. Green circle: HAE-A group. (E) Mantel Test is a correlation test to determine the correlation between two sets of distance measure matrices. And it is used to evaluate whether the sample distance in one matrix is related to the sample distance in the other matrix.