Effect of combined chronic predictable and unpredictable stress on depression-like symptoms in mice

Abstract

Background: Mental stress mainly induces depression, and predictable stress, as well as a constant bombardment of chronic unpredictable micro-stressors, always coexist in daily life. However, the combined effect of predictable and unpredictable stress on depression is still not fully understood.

Methods: The chronic restraint stress (CRS) is to restrain the mice for 6 h per day for 3 weeks, and the chronic unpredictable mild stress (CUMS) is to stimulate the mice with 7 different stressors for 3 weeks. We evaluated the combined effect of CRS and CUMS on depression-like symptoms using behavioral tests and investigated the action mechanism through analysis of neurotransmitters, brain-derived factors, inflammatory factors, antioxidants, and intestinal microorganisms.

Results: Our data suggested the combined stress of CRS and CUMS caused significant weight loss, food intake reduction, depression-like behaviors-including anhedonia, learned helplessness, and reduction in spontaneous activity-and even atrophy and severe structural damage to the hippocampus in mice. Our pathogenesis study showed that combined stress-induced the reduction of glucocorticoid receptor (GR) levels, loss of oligodendrocytes (NG2 and Olig2 cells), and inhibition of neuron proliferation in the CA1, CA3, and DG regions of the hippocampus, decreased the contents of monoamine neurotransmitters (5-HT and NE) and BDNF in the cerebral cortex, caused hyperactivity of the HPA system, led to immune dysfunction, aggravated oxidative stress, and weakened the capacity of antioxidants in mice. Compared with single stress, combined stress gave rise to a more significant diversity change of the gut microbiota.

Conclusions: Combined stress caused significant depression-like behaviors, atrophy, and severe structural damage to the hippocampus in mice via monoamine neurotransmitter, BDNF, HPA axis, neurogenesis, and neurodegenerative, immune, oxidative stress and gut-brain axis action pathways.

Keywords: Depression; chronic bondage stress; chronic unpredictability mild stress; combined stress; predictable stress.

Figure 1

The effect of stress stimulations on (A) body weight (B) sucrose preference ratio, (C) 2-hours food consumption, and (D) immobility time in the tail suspension test (TST) and forced swimming test (FST) of mice. Data are shown as mean ± SD (n=8). *, P<0.05; **, P<0.01 and ***, P<0.001 denote significant differences from the control group simultaneously; ^#, P<0.05 and ^##, P<0.01 denote a significant difference from the CS group simultaneously. The lowercase letters denote significant difference (P<0.05) from the corresponding group on Day 0 of each group. NS, not significant.

Figure 2

The effect of stress stimulations on depression-like behaviors of mice in the open-field test. (A) Rest time, (B) movement time, (C) central residence time, (D) center movement distance, (E) motion trajectory map of each group of mice in the OFT. Data are shown as mean ± SD (n=8). *, P<0.05, **, P<0.01 and ***, P<0.001 denote significant difference from the control group simultaneously; the lowercase letters denote significant difference (P<0.05) from the corresponding group on day 0 of each group. NS, not significant.

Figure 3

The effect of stress stimulations on biomarkers related to depression, immune factors, and oxidative stress of mice. (A) Monoamine neurotransmitter, (B) brain-derived neurotrophic factor (BDNF), (C) HPA axis correlation biomarker, (D) immune factors, (E) MDA, (F) antioxidases content of each group of mice. Data are shown as mean ± SD (n=8). *, P<0.05, **, P<0.01 and ***, P<0.001 denote significant difference from the control group; ^#, P<0.05 and ^##, P<0.01 denote a significant difference from the CS group.

Figure 4

The effect of stress stimulations on structure, expression of GR and oligodendrocytes, and neuron proliferation in key hippocampal subregions. (A) Coronal section image of the mouse brain, (B) immunofluorescence images of GR (red), (C) fluorescence intensity of GR, (D) immunofluorescence images of NG+ (red) and Olig2+ glial cells (green), (E) fluorescence intensity of NG2+ glial cells, (F) fluorescence intensity of Olig2+ glial cells, (G) immunofluorescence images of NeuN+ cells (green) and PCNA+ cells (red), (H) NeuN+ fluorescence intensity, (I) PCNA+ fluorescence intensity in subregions of the hippocampus. Data are shown as mean ± SD (n=8). *, P<0.05, **, P<0.01 and ***, P<0.001 denote significant difference from the control group; ^#, P<0.05, ^##, P<0.01 and ^###, P<0.001 denote significant difference from the control group.

Figure 5

PCoA of gut microbiota beta diversity with the (A) unweighted and (B) weighted UniFrac distances and (C) Bray-Curtis dissimilarity.

Figure 6

The effect of stress stimulations on the composition of the gut microbiota at the phylum level. (A) The relative abundances of phylum in the intestinal contents of mice after 3 weeks of stress treatment. The comparison of (B) CS group or (C) CUMS group with the control group on the relative abundances of Firmicutes, Verrucomicrobia, and Tenericutes in intestinal contents. Data are shown as mean ± SD (n=10). *, P<0.05 and **, P<0.01 denote significant difference from the control group.

Figure 7

The effect of stress stimulations on the composition of the gut microbiota at the order level. (A) The relative abundances of order in intestinal contents of mice 3 weeks after stress treatment. (B) The comparison of the CS group with the control group on the relative abundances of Clostridiales and Verrucomicrobia in intestinal contents. (C) The comparison of the CRS group with the control group on the relative abundance of Mycoplasmatales in intestinal contents. (D) The comparison of the CUMS group with the control group on the relative abundances of Lactobacillales and Mycoplasmatales in intestinal contents. Data are shown as mean ± SD (n=10). *, P<0.05 and **, P<0.01 denote significant difference from the control group.

Figure 8

The effect of stress stimulations on the composition of the gut microbiota at the family level. (A) The relative abundances of the family in intestinal contents of mice 3 weeks after stress treatment. (B) The comparison of the CS group with the control group on the relative abundances of Ruminococcaceae, Marinifilaceae, and Bacteroidaceae in intestinal contents. (C) The comparison of the CRS group with the control group on the relative abundance of Muribaculacaea in intestinal contents. (D) The comparison of the CUMS group with the control group on the relative abundances of Muribaculacaea and Lactobacillaceae in intestinal contents. Data are shown as mean ± SD (n=10). *, P<0.05 and **, P<0.01 denote a significant difference from the control group.

Figure 9

The effect of stress stimulations on the composition of the gut microbiota at the genus level. (A) The relative abundances of the genus in intestinal contents of mice 3 weeks after stress treatment. (B) The comparison of the CS group with the control group on the relative abundances of LachnospiraceaeNK4A136, Rikenella, Odoribacter, Bacteroides, Akkermansia, and Blautia in intestinal contents. (C) The comparison of the CRS group with the control group on the relative abundance of Rikenella and PrevotellaceaeUCG-001 in intestinal contents. (D) The comparison of the CUMS group with the control group on the relative abundances of Lactobacillus and Mycoplasma in intestinal contents. Data are shown as mean ± SD (n=10). *, P<0.05 and **, P<0.01 denote significant difference from the control group.

Figure 10

Analysis of species differences between groups. (A) Cladogram representing taxa with different abundances of gut microbiota at the baseline and (B) histogram of linear discriminant analysis scores computed for features differentially abundant among groups. The linear discriminant analysis (LDA) score on the log10 scale is showed at the bottom. The greater the LDA score is, the more significant the phylotype biomarker is in the comparison.