Temporal dynamics of pro-inflammatory cytokines and serum corticosterone following acute sleep fragmentation in male mice

Abstract

Obstructive sleep apnea is increasing worldwide, leading to disordered sleep patterns and inflammatory responses in brain and peripheral tissues that predispose individuals to chronic disease. Pro-inflammatory cytokines activate the inflammatory response and are normally regulated by glucocorticoids secreted from adrenal glands. However, the temporal dynamics of inflammatory responses and hypothalamic-pituitary-adrenal (HPA) axis activation in relation to acute sleep fragmentation (ASF) are undescribed. Male C57BL/6J mice were exposed to ASF or control conditions (no ASF) over specified intervals (1, 2, 6, or 24 h) and cytokine gene expression (IL-1β, TNF-α) in brain and peripheral tissues as well as serum glucocorticoid and interleukin-6 (IL-6) concentration were assessed. The HPA axis was rapidly activated, leading to elevated serum corticosterone from 1-24 h of ASF compared with controls. This activation was followed by elevated serum IL-6 concentration from 6-24 h of ASF. The tissue to first exhibit increased pro-inflammatory gene expression from ASF was heart (1 h of ASF). In contrast, pro-inflammatory gene expression was suppressed in hypothalamus from 1 h of ASF, but elevated at 6 h. Because the HPA axis was activated throughout ASF, this suggests that brain, but not peripheral, pro-inflammatory responses were rapidly inhibited by glucocorticoid immunosuppression.

Fig 1. Experimental protocol for acute sleep fragmentation (ASF) time-course study.

Mice were exposed to 1, 2, 6, 12, or 24 h of ASF, which involved a sweeping bar that moves horizontally across a modified cage every 120 s. Mice were acclimated to the sleep fragmentation chamber cage (no bar movement) for 48 h before starting the experiment. Control (CON; no sleep fragmentation) mice experienced no bar movement. ZT0 indicates when lights turned on; ZT12 indicates when lights turned off.

Fig 2. Duration of acute sleep fragmentation (ASF) alters baseline glucocorticoid and IL-6 levels in serum.

A) Corticosterone (cort) concentration in male mice subjected to acute SF (0, 1, 2, 6, 12, and 24 h of ASF or no SF (CON)). Samples sizes are n = 9–10 per group. B) IL-6 levels in male mice subjected to acute SF (0, 1, 2, 6, 12, and 24 h of ASF or no SF (CON). Samples sizes are N = 9-10/group. Significant effect of ASF (*** and **** denote p < 0.001 and 0.0001, respectively) relative to NSF at each time point was determined by two-way ANOVA followed by Bonferroni multiple comparisons post hoc tests. Differing lowercase and upper-case letters denote p < 0.05 for NSF and CON groups, respectively and were analyzed using a one-way ANOVA and Tukey’s HSD post hoc tests. Bar plots shown as means ± 1 SE and p was set at 0.05 for statistical significance.

Fig 3. Effects of sleep fragmentation, time, and their interaction on TNF-α and Il-1β gene expression in peripheral tissues.

Panels show TNF-α and Il-1β gene expression in spleen (A, B), heart (C, D), epididymal white adipose tissue (EWAT; E, F) and liver (G, H), respectively (n = 8-10/group, time-course: 0, 1, 2, 6, 12, 24h). Significant effect of sleep treatment (** denotes p < 0.01) at each time point was determined by two-way ANOVA followed by Bonferroni multiple comparisons post hoc tests. Differing lowercase and upper-case letters denote p < 0.05 across different time points for CON and ASF, respectively and were analyzed using a one-way ANOVA and Tukey’s HSD post hoc tests. Bar plots shown as means ± 1 SE and p was set at 0.05 for statistical significance.

Fig 4. Effects of sleep fragmentation, time, and their interaction on TNF-α and Il-1β gene expression in select brain regions.

Panels show TNF-α and Il-1β gene expression in HIP (A, B), PFC (C, D), and HYP (E, F), respectively (n = 9-10/group, Acute SF time-course: 0, 1, 2, 6, 12, 24h). Significant effect of SF (*, ** and *** denote p < 0.05, 0.01, and 0.001, respectively) was determined by two-way ANOVA followed by Bonferroni multiple comparisons post hoc test. Differing lowercase and upper-case letters denote p < 0.05 across different time points for CON and ASF, respectively and were analyzed using a one-way ANOVA and Tukey’s HSD post hoc tests. Bar plots shown as means ± 1 SE and p was set at 0.05 for statistical significance. HIP: Hippocampus; PFC: prefrontal cortex; HYP: Hypothalamus.