Sex Differences in the Impact of Shift Work Schedules on Pathological Outcomes in an Animal Model of Ischemic Stroke

Abstract

Circadian clock desynchronization has been implicated in the pathophysiology of cardiovascular disease and related risk factors (eg, obesity, diabetes). Thus, we examined the extent to which circadian desynchronization exacerbates ischemic stroke outcomes and whether its detrimental effects on stroke severity and functional impairments are further modified by biological sex. Circadian entrainment of activity rhythms in all male and female rats was observed during exposure to a fixed light-dark (LD) 12:12 cycle but was severely disrupted when this LD cycle was routinely shifted (12 h advance/5 d) for approximately 7 weeks. In contrast to the regular estrous cycles in fixed LD animals, cyclicity was abolished and persistent estrus was evident in all shifted LD females. The disruption of estrous cyclicity in shifted LD females was associated with a significant increase in serum estradiol levels relative to that observed in fixed LD controls. Circadian rhythm disruption exacerbated stroke outcomes in both shifted LD male and female rats and further amplified sex differences in stroke impairments. In males, but not females, circadian disruption after exposure to the shifted LD cycle was marked by high rates of mortality. In surviving females, circadian desynchronization after exposure to shifted LD cycles produced significant increases in stroke-induced infarct volume and sensorimotor deficits with corresponding decreases in serum IGF-1 levels. These results suggest that circadian rhythm disruption associated with shift work schedules or the irregular nature of our everyday work and/or social environments may interact with other nonmodifiable risk factors such as biological sex to modulate the pathological effects of stroke.

Figure 1.. Effects of shifted LD cycles on circadian entrainment and other properties of the rhythm in wheel-running activity.

A, 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 (12 h/5 d) LD 12:12 cycle (right). Actograms are plotted over a 24-hour 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. B, Rhythm amplitude, activity duration (α), and total daily wheel-running activity of adult male (top) and female (bottom) rats exposed to fixed or shifted LD cycles. Bars depict average wheel revolutions per 24 hours (±SEM). *, P < .05.

Figure 2.. Shifted LD cycles abolish estrous cyclicity and increase serum estradiol levels.

A, Determination of estrous cyclicity and cycle length in adult female rats exposed to fixed or shifted LD 12:12 cycles. Bars depict baseline determinations in all animals before experimental lighting conditions (left) and posttreatment analyses after exposure to fixed or shifted LD cycles for 2 months. The average (±SD) estrous cycle length is shown above the corresponding bar. B, Serum estradiol levels in female rats exposed to fixed or shifted LD cycles. Bars depict average estradiol levels (±SEM) in terminal blood obtained 5 days after MCAo surgery. *, P < .05.

Figure 3.. Shifted LD cycles dramatically increase mortality after MCAo-induced stroke in adult male but not female rats.

Kaplan-Meier survival plots after MCAo-induced stroke in adult male (left panel) and female (right panel) rats exposed to fixed or shifted LD 12:12 cycles. The individual plots depict the conditional probability of survival over postsurgery days 0–5. In males, but not females, exposure to shifted LD cycles had significant effects in increasing mortality (P < .005) and decreasing survival time relative to that found in fixed LD controls.

Figure 4.. Shifted LD cycles amplify the size of MCAo-induced infarction in adult female rats.

TTC staining was used to quantify MCAo-induced infarct volume in adult female rats exposed to fixed (n = 18) or shifted (n = 18) LD 12:12 cycles. A, Representative TTC-stained sections illustrating cortical and striatal lesions (unstained) in the left hemisphere. B, Bars depict infarct volumes (cortex and striatum) normalized to the contralateral hemisphere. *, P < .05.

Figure 5.. Shifted LD cycles exacerbate MCAo-induced impairment on the adhesive tape test in adult female rats.

Sensorimotor performance in female rats exposed to fixed or shifted LD cycles was assessed 2 days before (PRE) and 5 days after (POST) MCAo surgery using the adhesive tape test. Individual panels depict prestroke response latency for tape removal (left panel) and differences between post- and prestroke latency times (right panel) on the contralesional (right) side. Bars represent average values (±SEM). *, P < .05.

Figure 6.. MCAo-induced impairment on the vibrissae-evoked forelimb placement task is similar in adult female rats exposed to fixed or shifted LD cycles.

Sensorimotor performance in female rats exposed to fixed or shifted LD cycles was examined 2 days before (PRE) and 5 days after (POST) MCAo surgery using the vibrissae evoked forelimb placement task. Bars depict percent correct responses for the ipsilesional (IPSI) and contralesional (CONTRA) paw before and after stroke. *, P < .05.

Figure 7.. Shifted LD cycles decrease serum IGF-1 levels in adult female rats.

Serum IGF-1 levels in female rats exposed to fixed or shifted LD cycles. Bars depict average IGF-1 levels (±SEM) in terminal blood obtained 5 days after MCAo surgery. *, P < .05.