IL-4/10 prevents stress vulnerability following imipramine discontinuation

Abstract

Background: Identifying stress vulnerability after antidepressant discontinuation may be useful in treating relapses in depression. Previous studies have suggested significant effects of the immune system as well as the central nervous system (CNS) on progression and induction of major depression. In the present study, we hypothesized that the factors that are not rescued by a tricyclic antidepressant imipramine may be associated with stress vulnerability and relapses in depression.

Methods: To address this issue, mice were exposed to 2 h of restraint stress for 21 consecutive days (chronic restraint stress (CRS)) with or without co-treatment of imipramine. These groups were exposed to an electronic foot shock (FS) as additional stress after imipramine washout. Main targets of stress and antidepressants were analyzed in the hippocampus, lymph node, and serum after a series of depression-like behavior analysis.

Results: In this study, we found for the first time that mice exposed to CRS with a tricyclic antidepressant imipramine co-treatment, which did not show depressive-like behaviors, were vulnerable to subsequent stressful stimuli compared to the non-stressed mice after imipramine washout. CRS mice with imipramine co-treatment did not show any difference in BDNF, serotonin receptors, pro-inflammatory cytokines, or kynurenine pathway in the hippocampus compared to the controls. However, peripheral IL-4, IL-10, and alternatively activated microglial phenotypes in the hippocampus were not restored with sustained reduction in CRS mice despite chronic imipramine administration. Supplementing recombinant IL-4 and IL-10 in co-Imi+CRS mice prevented the stress vulnerability on additional stress and restored factors related to alternatively activated microglia (M2) in the hippocampus.

Conclusion: Thus, our results suggest that the reduced IL-4 and IL-10 levels in serum with hippocampal M2 markers may be involved in the stress vulnerability after imipramine discontinuation, and the restoration and modulation of these factors may be useful to some forms of depression-associated conditions.

Fig. 1

Chronic restraint stress-induced anxiety-like and depressive-like behaviors and imipramine co-treatment prevented the induction of stress-related behaviors. Mice were exposed to restraint stress for 21 days (CRS) with saline or imipramine (20 mg/kg; co-Imi+CRS). A serial of behavioral assessments were finished within a week after the termination of CRS and imipramine co-treatment. Anxiety-like behaviors, such as light–dark (LD) box and elevated plus maze (EPM), were assessed using EthoVision XT9 (a). LD the time spent in the dark zone during the 10 min was measured (b). EPM the time spent in the open arms was measured and was expressed as a percentage (c). Sucrose preference (SP) for 1 % sucrose solution over regular drinking water was examined for 2 days after 2 days of inhabitation to two bottle conditions (d). Tail suspension test (TST) was done for a 7-min period, and the duration that the subject remained immobile was calculated (e). Forced swimming test (FST) was done for 5 min following a minute of pre-swimming, and the total immobility time after the pre-swimming was measured (f). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the controls unless indicated otherwise with an arrow, n = 10–13 in each group and the data shown are mean ± standard mean error (SEM). Serum glucocorticoid level (Gc level) was analyzed immediately after CRS by performing enzyme-linked immunosorbent (EIA) assay (g). **p < 0.01 compared with the controls, n = 5 in each group and the data shown are mean ± standard mean error (SEM)

Fig. 2

CRS mice with imipramine co-treatment exhibited anxiety-like and depressive-like behaviors upon an exposure to short-term acute stress. Drug treatment and stressful stimuli exposure timeline: CRS, chronic restraint stress; SP, sucrose preference test; EPM, elevated plus maze; TST, tail suspension test; RS, restraint stress; LD, light–dark box; FST, forced swimming test (a). Depressogenic effect of foot shock stress was assessed, and foot shock stress alone does not affect anxiety-like or depressive-like behaviors in the non-stressed (CON) and CRS mice (b). Prior to the exposure to foot shock, CRS mice with imipramine co-treatment (co-Imi+CRS) did not exhibit anhedonic behavior (sucrose preference test, SP), anxiety-like behaviors in EPM, and depressive-like behaviors in TST compared with CON (c). However, following the exposure to electric foot shock, they (co-Imi+CRS) showed decreased sucrose preference, exhibited anxiety-like behavior in LD, and depressive-like behaviors in FST compared with CON (d). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the controls unless indicated otherwise with an arrow, n = 8–10 in each group and the data shown are mean ± standard mean error (SEM). Three times independent experiments was performed, giving a total of nine per group in the final analysis for Western blot

Fig. 3

Depression-related factors in the hippocampus were normalized by imipramine co-treatment while M2-related microglia phenotypes were not rescued by imipramine. CRS was conducted for 2 h/day for 21 consecutive days by placing the mouse in a 50-mL Corning tube, and 20 mg/kg of imipramine was administered intraperitoneally 30 min prior stress. The mice were sacrificed 1 day after Fig. 1 of behavior assessment. The hippocampi were dissected for quantitative reverse transcriptase polymerase chain reactions (a, c) and Western blot (b) analysis. Quantitative reverse transcriptase polymerase chain reactions were done using mRNA extracted from the mouse hippocampus (n = 10–15). The CT values were normalized as a ratio as controls being 1, which is indicated by dotted lines (a). Western blot analysis was done using 50 μg of total protein extracted from the mouse hippocampus (n = 3). Expressed BDNF, serotonin receptor 1A (5-HT _1A), serotonin receptor 2A (5-HT _2A), IDO, CX3CR1, and β-actin were quantified using MultiGauge application (Fujifilm) (b). Changes in mRNA of NADPH oxidase (NOX2) and M2 microglia markers, such as fractalkine receptor (CX3CR1), CD200 receptor (CD200R), and CD206, were measured (n = 7–10). RQ value refers to the ratio of respective factors as a percentage of the controls (c). The data shown are mean ± standard mean error (SEM) *p < 0.05, ***p < 0.001 compared with the controls unless indicated otherwise with an arrow

Fig. 4

The reduced IL-4 and IL-10 with Th2- and T_reg-related transcription factors were not rescued by imipramine treatment. CRS was conducted for 2 h/day for 21 consecutive days by placing the mouse in a 50-mL Corning tube, and 20 mg/kg of imipramine was administered intraperitoneally 30 min prior stress. The mice were sacrificed 1 day after Fig. 1 of behavior assessment. The mesenteric lymph nodes (a) were dissected for quantitative reverse transcriptase polymerase chain reactions and serum (b) was obtained to measure circulating levels of pro-and anti-inflammatory cytokines using Bio-Rad Bio-Plex^® assay. The CT values were normalized as a ratio as controls being 1, which is indicated by dotted lines. RQ value refers to the ratio of respective transcription factors as a percentage of the controls. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the controls, n = 10–15 per group and the data shown are mean ± standard mean error (SEM)

Fig. 5

Supplement of IL-4 and IL-10 prevents stress vulnerability following imipramine discontinuation with restoration of type 2 microglia marker messenger RNAs. A combination of IL-4 (100 ng) and IL-10 (100 ng) for 5 days was treated intraperitoneally in non-stressed mice (controls) and CRS mice. A combination of IL-4 (100 ng) and IL-10 (100 ng) for 5 days itself does not have an antidepressant-like effect in controls or CRS mice (a). The combination of IL-4 and IL-10 for 5 days does not alter the depression-associated factors, BDNF, and M2-related microglia factor, CX3CR1, in the hippocampus (b). Effect of IL-4 and IL-10 in preventing stress vulnerability and depressive-like behaviors of co-Imi+CRS mice was assessed using sucrose preference (SP) test, elevated plus maze (EPM), tail suspension test (TST), and forced swimming test (FST) (c). Changes in M2 microglia marker expressions, such as fractalkine receptor (CX3CR1), CD200 receptor (CD200R), and CD206, were measured 1 day after (c) of behavior assays (d). RQ value refers to the ratio of respective factors as a percentage of the controls. +p < 0.05, +++p < 0.001 compared with the groups indicated by an arrow. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the controls, n = 12–15 per group and the data shown are mean ± standard mean error (SEM)