Social isolation induces neuroinflammation and microglia overactivation, while dihydromyricetin prevents and improves them

Abstract

Background: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country's mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system.

Methods: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured.

Results: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes.

Conclusion: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety.

Keywords: Anxiety; Dihydromyricetin (DHM); GABAAR; Microglia; Neuroinflammation; Social isolation.

Fig. 1

DHM reduces social isolation-induced anxiety. A Effects of social isolation and treatment with DHM on anxiety-like behaviors as measured by the time (min) spent in the open and closed arms of the elevated plus-maze. B Effects of social isolation and treatment with DHM on locomotor activity, exploratory behaviors as measured by running distance (total distance of moving), rearing (total number of times of rearings), corner (the total duration the mouse stayed in the 4-corner 10 × 10 cm squares), and center time (the total time duration the mouse stayed in the center 20 × 20 cm square), in the open field assay. One-way ANOVA followed by multiple comparison, Holm–Sidak method to the control. For running length, p < 0.001; for numbers of rearings, p  < 0.001. For stay in corners p  < 0.001. For stay in the center, p  < 0.001. * = p  ≤ 0.05 vs. vehicle group housing control (G2 + Veh2). (n = 10–11/group)

Fig. 2

Changes in gephyrin protein expression after social isolation and the effect of DHM treatment. Grouped-house (G4 + Veh2), single-housed mice (Iso4 + Veh2), and single-housed with DHM treatment (Iso4 + D2). β-Actin from the same blot was used as a loading control. One-way ANOVA followed by multiple comparisons, Holm–Sidak’s method; * = p ≤ 0.05 vs. group housing control (G4 + Veh2), n = 5/group

Fig. 3

The effects of social isolation-induced anxiety and DHM treatment on microglia activation and proliferation in the hippocampal CA area. A Representative images showing the effect of social isolation-induced anxiety on the number of labeled microglia in the hippocampus, Iba-1 (red), DAPI (blue). B Confocal single-cell microglia images for the G2 + Veh2, Iso + Veh2, and Iso + D2 obtained using a 63X oil-immersion objective. C Quantification analysis of the microglia number in CA1 and CA2 area, data presented as the number of cells per 1 mm². One-way ANOVA followed by Sidak multiple comparisons test was used for statistical analysis. Each point represents cells number in 2–3 individual sections from n = 5 mice, values represented as mean ± SEM, * = p ≤ 0.05, n = 5

Fig. 4

DHM treatment modulates microglia morphology in the CA1 and CA2 area of hippocampus. A Representative photos of the hippocampus CA1 where the white rectangular are regions for photomicrograph analysis, stained for DAPI (blue, left panel) and Iba-1 (red, right panel) from the control group. B Illustration for the box-counting method used for lacunarity calculation. C representative images for binary microglial and convex hull (green) and enclosing circle (pink) that were used to calculate density, perimeter, and maximum span across the hull, and the corresponding microglial in each group. D Microglial morphology profile illustrated in lacunarity, cell perimeter (E), density (cell area/convex hull area) (F), and the maximum span across the convex hull (G). One-way ANOVA followed by Sidak multiple comparisons test was used for statistical analysis. Each point represents individual microglia 3–4 cells from n = 5 mice, values represented as mean ± SEM, * = p ≤ 0.05

Fig. 5

The effect of DHM on the levels of serum corticosterone, p-NF-kB p65 protein expression, and proinflammatory cytokines expression after social isolation. A1 Representative Western blots of phospho-NF-kB p65 (65 kDa), NF-kB p65 (65 kDa), and β -actin (42 kDa). A2 and A3 Quantitative analysis ratio for the protein expression of phospho-NF-κB p65 and NF-κB p65 with the loading control β-actin F (4, 10) = 1.485 p = 0.5885. B Serum level of corticosterone (ng/ml) after social isolation (Iso2 + Veh2) and following DHM treatment 2 mg/kg. One-way ANOVA followed by multiple comparisons, Holm–Sidak’s method. C Heat map of serum cytokines and chemokines level. The color key indicates response level ranging from the highest (red) to the lowest (pink). Data represented as the inverted mean gray value expressed in each cytokine/chemokine. Each data point was normalized to the baseline value (G2 + Veh2). (G2 + Veh) grouped housed vehicle, (G2 + D2) grouped housed DHM, (Iso2 + Veh2) socially isolated vehicle, (Iso2 + D2) socially isolated DHM, n = 4–5 per group. * = p ≤ 0.05 vs. group housing control (G2 + Veh2), n = 5/group

Fig. 6

Schematic summary of the various pathways involved in social isolation-induced neuroinflammation and anxiety-like behaviors. Social isolation (SI) induces stress that triggers disruption in the hypothalamus–pituitary–adrenal axis (HPA), increase in the levels of corticosterone (CORT), and overactivity in the sympathetic nervous system (SNS), which in turn might trigger NF-κB pathway activation and enhance the level of proinflammatory cytokines. Social isolation induces a reduction in pre-, post-, and/or extra-synaptic GABA_ARs, and in gephyrin expression leading to impairment in GABAergic neurotransmission. GABA_ARs are also expressed in microglia and astrocytes. Proinflammatory cytokines and GABA_AR disruption provokes microglia and astrocytes activation leading to further neuroinflammation damages and anxiety-like behaviors. DHM is a positive modulator of GABA_AR that plays a role in counteracting neuroinflammation and contribute to the anxiolytics effect