Preclinical evaluation of circadian rhythm in ischemic stroke outcomes

Abstract

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).

Keywords: Cerebral blow flow; Circadian rhythm; Neurological deficit; Stroke.

Figure 1.

The schematic diagram of the experimental design

Figure 2.

Corner test were performed to count right and left turn after stroke at 48hr of acute stroke. Right turn represents the right turn preference in mice after stroke. Right turn preference was used to measure the sensorimotor function which eventually impaired by stroke pathology at ipsilateral side (n=10–12 mice/group) ANOVA=p<0.0018 (F-test: 7.53). †Homogeneity of Variance was violated (F=7.91, p=0.0003), Welch’s ANOVA was performed.

Figure.3:

The infarct volume was observed by TTC staining of brain slices from each time points of stroke induction. The viable tissue was stained red color by TTC staining, but the infarcted/dead area remains unstained, exhibiting pale yellow/white color. Quantitative evaluation of percent infarction volume in different cohorts of mice was observed at 48h after MCAO. ‡ Homogeneity of Variance was verified (F=1.43, 0.2511).