Sex differences in the diathetic effects of shift work schedules on circulating cytokine levels and pathological outcomes of ischemic stroke during middle age

Abstract

Shift work is associated with increased risk for vascular disease, including stroke- and cardiovascular-related mortality. However, evidence from these studies is inadequate to distinguish the effect of altered circadian rhythms in isolation from other risk factors for stroke associated with shift work (e.g., smoking, poor diet, lower socioeconomic status). Thus, the present study examined the diathetic effects of exposure to shifted LD cycles during early adulthood on circadian rhythmicity, inflammatory signaling and ischemic stroke pathology during middle age, when stroke risk is high and outcomes are more severe. Entrainment of circadian activity was stable in all animals maintained on a fixed light:dark 12:12 cycle but was severely disrupted during exposure to shifted LD cycles (12hr advance/5d). Following treatment, circadian entrainment in the shifted LD group was distinguished by increased daytime activity and decreased rhythm amplitude that persisted into middle-age. Circadian rhythm desynchronization in shifted LD males and females was accompanied by significant elevations in circulating levels of the inflammatory cytokine IL-17A and gut-derived inflammatory mediator lipopolysaccharide (LPS) during the post-treatment period. Middle-cerebral artery occlusion, 3 months after exposure to shifted LD cycles, resulted in greater post-stroke mortality in shifted LD females. In surviving subjects, sensorimotor performance, assessed 2- and 5-days post-stroke, was impaired in males of both treatment groups, whereas in females, recovery of function was observed in fixed but not shifted LD rats. Overall, these results indicate that early exposure to shifted LD cycles promotes an inflammatory phenotype that amplifies stroke impairments, specifically in females, later in life.

Keywords: Circadian rhythm dysregulation; IL-17A; Inflammation; Lipopolysaccharide; Middle cerebral artery; Stroke pathology.

Fig. 1

Experimental Design. At 5-months of age, male and female rats were exposed for ≈3 months to fixed or shifted (12hr advance/5d) LD 12:12 cycles. Following this treatment period, animals (8-months old) in both groups were exposed to the same standard LD 12:12 cycle for 3 additional months and then subjected to experimental ischemic stroke surgery (at ≈11 months of age). Blood was collected from all animals: immediately before and after experimental LD cycle manipulations as indicated by the red arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Circadian patterns of wheel-running behavior in adult female and male rats exposed to fixed or shifted LD cycles. Representative records of wheel-running activity in adult male (top panel) and female (bottom panel) rats that were maintained in a fixed LD 12:12 cycle (left) or exposed to a shifted (12hr/5d) LD 12:12 cycle (right). Actograms are plotted over a 24-h period. The open and closed bars at the top respectively signify the timing of the light and dark phase in the fixed and shifted LD 12:12 cycles. Arrows on the right denote the interval when exposure to the shifted LD cycles was initiated (“treatment” phase) and when shifted LD animals were returned to the same regular LD 12:12 schedule as the fixed LD group (post-treatment phase).

Fig. 3

Effects of experimental LD cycles on circadian entrainment and other properties of the rhythm in wheel-running activity. (A) Rhythm amplitude and (B) total daily wheel-running activity (wheel revolutions/24hr) of adult male (top) and female (bottom) rats: during LD “treatment” (fixed, shifted) phase (TX; day 30–102), and the subsequent post-treatment phase (Post-TX; day 120–192) when both groups were exposed to the same fixed LD cycle. (C) Daytime activity (light counts/day) in male (top) and female (bottom) rats following exposure to fixed or shifted LD cycles (Post-TX; day 120–192). Bars depict mean values (+SEM). Asterisks indicate significant differences (p < 0.05) between the fixed and shifted LD groups in the amplitude of the rhythm in wheel-running activity.

Fig. 4

Effects of experimental LD cycles on body weight. Graphs depict weekly determinations (mean ± SEM) of body weight in adult male (top) and female (bottom) rats during exposure to fixed () or shifted () LD 12:12 cycles and immediately prior to ET-1-induced MCAo surgery. Simple linear regression analysis of LD treatment group differences indicates weight gain was significantly greater in fixed, than in shifted, LD females (*p = 0.03).

Fig. 5

Shifted LD cycles alter circulating levels of the inflammatory cytokine IL-17A and lipopolysaccharide (LPS). Serum levels (pg/ml) of IL-17A (A) and LPS (B) in male (left) and female (right) rats exposed to fixed or shifted LD cycles. Histograms depict mean IL-17A and LPS levels (+SEM) in saphenous blood collected immediately before (PRE) and after (POST) experimental LD cycle manipulations. *, p < 0.05; **, p < 0.01.

Fig. 6

Effects of experimental LD cycles on MCAo-induced mortality and infarct size. (A) Kaplan-Meier survival plots following MCAo-induced stroke in adult (female left panel) and male (right panel) rats exposed to fixed or shifted LD 12:12 cycles. The individual plots depict the conditional probability of survival over post-surgery days 0–5. Asterisk indicates the rate of mortality was significantly (p < 0.05) increased in females relative to that found in males in the shifted LD group. (B) Representative TTC-stained sections (top) illustrating MCAo-induced cortical and striatal infarcts (pale, unstained) in the left hemisphere of adult male (left) and female (right) rats exposed to fixed (n = 18) or shifted (n = 18) LD 12:12 cycles. The approximate borders of infarcted tissue are indicated by the dotted black outline on each section. Bar graphs (top) depict infarct volumes (cortex and striatum) normalized to the contralateral hemisphere.

Fig. 7

MCAo-induced impairment of sensorimotor performance in adult female and male rats exposed to fixed or shifted LD cycles. Sensorimotor performance was examined 1 day before (PRE) and at 2 days (2d) and 5 days (5d) after MCAo surgery using the (A) vibrissae evoked forelimb placement task (VIB) and (B) adhesive tape test (ART) in male (left) and female (right) rats exposed to fixed or shifted LD cycles. Histograms depict average values (+SEM) for percent correct responses for the ipsi-lesional (IPSI) and contra-lesional (CONTRA) paw before and after stroke (A) and for latency in seconds to remove tape from the forepaw (B). *, p < 0.05 (PRE vs 2d; PRE vs 5d).