Time Dimension Influences Severity of Stroke and Heightened Immune Response in Mice.

Ischemic stroke is caused by obstructed cerebral blood flow, which results in neurological injury and poor outcomes. Pro-inflammatory signaling from both residential and infiltrating immune cells potentiates cerebral injury and worsens patient outcomes after stroke. While the occurrence of a stroke exhibits a time-of-day-dependent pattern, it remains unclear whether disrupted circadian rhythms modulate post-stroke immunity. In this study, we hypothesized that stroke timing differentially affects immune activation in mice. Following middle cerebral artery occlusion (MCAO), circadian genes BMAL1, CLOCK, Cry1, and Cry2 elevated at ZT06, with a significant difference between ZT06 and ZT18. Conversely, expression of the negative limb circadian clock gene, Per1, decreased at ZT06 and ZT18 in stroke mice compared to sham. Paralleling these circadian gene expression changes, we observed a significant increase in TNF-α and a decrease in IL-10 expression at 48 h post-MCAO, when the procedure was performed at ZT06 (MCAO-ZT6), which corresponds to a deep sleep period, as compared to when the stroke was induced at ZT12 (MCAO-ZT12), ZT18 (MCAO-ZT18), or ZT0 (MCAO-ZT12). Similarly, increased pro-inflammatory, decreased anti-inflammatory monocytes, and increased NLRP3 were observed in blood, while changes in the expression of CD11b and Iba1 were noted within brain tissue at 48 h of MCAO-ZT06, as compared to MCAO-ZT18. Consistent with the increased immune response, infarct volume and sensorimotor deficits were greater in MCAO-ZT06 mice compared to MCAO-ZT18 mice at 48 h. Finally, we found reduced weight and length of the spleen while splenocytes showed significant time-dependent changes in Tregs, Bregs, and monocytes in MCAO-ZT06 mice. Taken together, this study demonstrates that circulating and splenic immune responses following ischemic stroke exhibit a circadian expression pattern which may contribute to time-of-day-dependent stroke outcomes.

Preclinical evaluation of circadian rhythm in ischemic stroke outcomes.

Stroke is a leading cause of disability and death worldwide. There is evidence that there is a circadian rhythm in stroke with peak occurrence in the morning (6 to 10 am). However, it is not clear if the size of infarcts and the outcome of stroke also varies during the 24-hour period. We hypothesized that the size of cerebral infarct and outcome from stroke would show circadian variation in a mouse suture occlusion model. Seven to eight-month-old C57BL/6J (n =10-12 mice/group) mice were randomly assigned to undergo middle cerebral artery occlusion (MCAO) for 60 minutes at different time points during the 24h day following zeitgeber time at ZT0, ZT6, ZT12, and Z18. Cerebral blood flow was monitored by Laser Speckle Contrast Imaging at baseline after occlusion, and again at 24h post-occlusion. Neurological deficit was observed by using Bederson score at 24h and 48h. The corner test was used to detect unilateral abnormalities in sensory and motor functions in the stroke mice at 48h. To estimate brain infarction, 2,3,5-tryphenyltetrazolium chloride staining was performed 48h after stroke and the infarct area was quantified using NIH-Image J software. We did not find a significant difference in cerebral blood flow at any time point. There was a significant decrease in neurological deficit as assessed using the Bederson Score from 24h (1.82 ± 1.11) to 48h (1.10 ± 0.12) in the ZT18 (midnight) period (p = 0.0025), however there were no differences between groups at 48h. In the corner test, we found right turn preference significantly higher (p = 0.0348) at noon/ZT06 (9.5 ± 1.06) compared to the fully awake (5.5 ± 4.06) (midnight, ZT18) period and ZT0 (6 am, 4.8 ± 0.97, p = 0.0087). Similarly, the infarction volume was significantly higher (p = 0.0220) during the sleep (ZT06, noon) period (35.22 ± 20.77) than when the ischemic mice were fully awake during the midnight/ZT18 period (15.68 ± 7.54). This is the first report demonstrating that mice have larger infarcts and worse short-term outcomes during their sleep period (noon/ZT06) than during their awake period (midnight/ZT18).

Obesity alters the peripheral circadian clock in the aorta and microcirculation.

OBJECTIVE: Perturbation of daily rhythm increases cardiovascular risk. The aim of this study was to determine whether obesity alters circadian gene expression and microvascular function in lean mice and obese (db/db) mice. METHODS: Mice were subjected to normal LD or DD to alter circadian rhythm. Metabolic parameters and microvascular vasoreactivity were evaluated. Array studies were conducted in the am and pm cycles to assess the rhythmicity of the entire genomics. Rhythmic expression of specific clock genes (Bmal1, Clock, Npas2, Per1, Per2, and Cry1), clock output genes (dbp), and vascular relaxation-related genes (eNOS, GTPCH1) were assessed. RESULTS: Obesity was associated with metabolic dysfunction and impaired endothelial dilation in the microvasculature. Circadian rhythm of gene expression was suppressed 80% in both macro- and microcirculations of obese mice. Circadian disruption with DD increased fasting serum glucose and HbA1c in obese but not lean mice. Endothelium-dependent dilation was attenuated in obese mice and in lean mice subjected to DD. Rhythmic expression of per1 and dbp was depressed in obesity. Expression of eNOS expression was suppressed and GTPCH1 lost rhythmic expression both in obesity and by constant darkness. CONCLUSION: These results suggest that obesity reduces circadian gene expression in concert with impaired endothelial function. The causal relationship remains to be determined.